Cell penetrating peptides

ABSTRACT

The present invention relates to peptides, in particular cell penetrating peptides, of 40 amino acid residues or less comprising at least one directly glycosylated amino residue and one or more arginine rich arm domains, and to conjugates of such cell penetrating peptides with a therapeutic molecule. The present invention further relates to the use of the peptides or conjugates in methods of treatment or as a medicament, especially in the treatment of genetic disorders of the central nervous system. page.

TECHNICAL FIELD

The present invention relates to peptides, in particular cellpenetrating peptides, and to conjugates of such cell penetratingpeptides with a therapeutic molecule. The present invention furtherrelates to use of such peptides or conjugates in methods of treatment oras a medicament, especially in the treatment of genetic disorders of thecentral nervous system.

BACKGROUND

Disruption of alternative splicing underlies many diseases, andmodulation of splicing using antisense oligonucleotides can havetherapeutic implications. Splice-switching antisense oligonucleotides(SSOs) are emerging treatments for neuromuscular diseases, with severalSSOs currently undergoing clinical trials for conditions such as spinalmuscular atrophy (SMA) and Duchenne muscular dystrophy (DMD), whereantisense-mediated exon skipping can restore the open reading frame andallow the synthesis of partly functional proteins instead ofnon-functional ones.

However, therapeutic development of these promising antisensetherapeutics has been hampered by poor tissue penetration and cellularuptake. This is especially the case where crossing of the blood-brainbarrier (BBB) to reach targets in the central nervous system (CNS) isrequired.

In SMA, for example, therapeutic SSOs can be used to target theregulatory elements of splicing. One such target is the intronic splicesilencer N1 (ISS-N1) site within intron 7 of the SMN2 gene. SSOtargeting of this site enhances exon 7 inclusion to generate a fullyfunctional full length SMN2 (FLSMN2) mRNA and hence up-regulates SMNprotein production (1,2). One such 2′-O-methexyethyl phosphorothioateSSO is being developed by Ionis Pharmaceuticals and Biogen for thetreatment of SMA in infants and children, which has progressed to phaseIII clinical trials after promising data from open label phase IIstudies (3). This drug (Nusinersen) has just been approved for clinicaluse by the FDA in the USA.

However, currently this type of SSO therapeutic is not able to cross theblood-brain barrier (BBB) to reach the target motor neurons, thereforedelivery of the therapeutic must be via intrathecal injection to obtainbroad CNS distribution. In addition, the currently available SSOtherapeutics are inefficient and require high doses in order to effectany physiological changes.

Furthermore, it has become recognized that restoration of the SMN2 geneis also essential in the peripheral muscles in addition to within thebrain and spinal cord compartments in order for long term rescue of aseverely affected SMA mouse (4). SSO therapeutic delivery must accountfor both of these needs.

Thus there is a strong impetus for the development of efficient deliverysystems for therapeutic SSOs both to facilitate entry into brain andspinal cord via the circulation and to penetrate target cells better inboth periphery and in the CNS, as well as to enhance the efficiency ofthe therapeutic SSOs in order to reduce drug doses.

The use of viruses as delivery vehicles has been suggested, however thisis limited due to the immunotoxicity of the viral coat protein andpotential oncogenic effects (5). Alternatively, a range of non-viral CNSdelivery vectors have been developed, amongst which peptides have shownthe most promise due to their small size, low toxicity, targetingspecificity and ability of trans-capillary delivery of large bio-cargoes(6-8). Several peptides have been reported for their ability to permeatecells either alone or carrying a bio-cargo (6, 7).

For several years, cell-penetrating peptides (CPPs) have been used asconjugates of certain types of SSOs (in particular charge neutralphosphorodiamidate morpholino oligomer (PMO) and peptide nucleic acids(PNA)) to enhance their cell delivery by effectively carrying themacross cell membranes to reach their pre-mRNA target sites in the cellnucleus. It has been shown that PMO therapeutics conjugated to certainarginine-rich CPPs (known as P-PMOs) can enhance dystrophin productionin skeletal muscles following systemic administration in a mdx mousemodel of DMD (9-11).

In particular, arginine-rich CPPs known as PNA/PMO internalizationpeptides (Pips), comprised of two arginine-rich sequences separated by acentral short hydrophobic sequence have been developed. These ‘Pip’peptides were designed to improve serum stability whilst maintaining ahigh level of exon skipping, initially by attachment to a PNA cargo(12). Further derivatives of these peptides were designed as conjugatesof PMOs, which were shown to lead to high body-wide skeletal muscledystrophin production, and importantly also including the heart,following systemic administration (13).

More recently, promising results have been obtained from the use of aparticular ‘Pip’ peptide conjugate for the treatment of SMA: Pip6a-PMO.This peptide conjugate was administered by systemic delivery into adult,non-affected mice that maintain a copy of the human SMN2 transgene andthe production of full length SMN2 transcript was measured byquantitative polymerase chain reaction (qPCR). This experiment showedthat use of a Pip6a-PMO conjugate was able to generate significant exoninclusion of SMN2 exon 7 both in skeletal muscles as well as in thebrain and spinal cord. A greater than 30% increase in full-length SMN2transcripts was observed in all areas of the brain, spinal cord, andskeletal muscles (14).

However, this Pip′-PMO conjugate, when systemically injected into mice,requires a high dose which results in insufficient tolerability from themice and therefore does not provide a large enough dosing range fortherapeutic development (14).

The present inventors have now identified, synthesized and tested anumber of cell penetrating peptides according to the present invention.These peptides maintain good levels of cell penetration as well astissue penetration, in skeletal muscles as well as in compartments ofthe brain and spinal cord. This action of these peptides further givesrise to enhanced levels of exon inclusion in both skeletal muscles aswell as in compartments of the brain and spinal cord relative topreviously available carrier peptides.

In addition, the present peptides show a shorter recovery time followingsystemic injection into mice, indicating that the peptides of theinvention may be more applicable as a therapeutic treatment for humansthan previous cell penetrating peptides. A shorter recovery time isadvantageous in having minimal disruption to patient's everyday livesand increasing quality of life. It also reduces the burden on healthcarefacilities in accommodating patients whilst they are treated.Furthermore, the present peptides are demonstrated to show reducedtoxicity following similar systemic injection into mice when comparedwith previous cell penetrating peptides. This also indicates that thepeptides of the invention are more suitable for use as a therapy forhumans that previously available peptides. Reduced toxicity means thatthe peptides can be used safely as a therapy.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there is provideda peptide comprising at least one directly glycosylated amino acidresidue and one or more arginine-rich arm domains, wherein the totallength of the peptide is 40 amino acid residues or less.

According to a second aspect of the present invention, there is provideda conjugate comprising the peptide of the first aspect covalently linkedto a therapeutic molecule.

According to a third aspect of the present invention, there is provideda pharmaceutical composition comprising the conjugate of the secondaspect.

According to a fourth aspect of the present invention, there is provideda conjugate according to the second aspect for use as a medicament.

According to a fifth aspect of the present invention, there is provideda method of treating a disease in a subject comprising administering theconjugate of the second aspect to the subject in a therapeuticallyeffective amount.

According to a sixth aspect of the present invention, there is providedan isolated nucleic acid encoding the peptide of the first aspect or theconjugate of the second aspect.

According to an seventh aspect of the present invention, there isprovided an expression vector comprising the nucleic acid sequence ofthe sixth aspect.

According to an eighth aspect of the present invention, there isprovided a host cell comprising the expression vector of the seventhaspect.

DETAILED DESCRIPTION

The inventors have produced a series of peptides that are suitable foruse as carrier peptides to deliver therapeutic molecules into cells.These peptides of the invention are particularly suited to delivery oftherapeutic molecules into cells of the central nervous system inaddition to delivery of therapeutic molecules into cells in peripheraltissues.

Surprisingly, the inventors have discovered that the presence of atleast one directly glycosylated amino acid residue in combination withshort arginine-rich arm sequences produces peptides which, whendelivered as a conjugate with an antisense oligonucleotide therapeuticinto cells, or when administered in vivo, provide enhanced cellpenetration in skeletal muscles, as well as enhanced penetration intomany compartments of the brain and spinal cord compared with currentlyavailable cell penetrating peptides.

In the context of the disease SMA, enhanced cell penetration by peptidesof the invention linked to a suitable therapeutic molecule can be shownby increased exon inclusion, which causes cells to produce more of thefull length SMN2 gene transcript instead of the truncated version. Thepresence of more full length SMN2 enables some level of muscle functionto be restored in individuals suffering from this disease. The peptidesof the present invention, when used as a conjugate with an antisenseoligonucleotide therapeutic designed to target the SMN2 gene, are shownherein to have significantly increased cell penetration when comparedwith currently available peptides conjugate to the same antisenseoligonucleotide therapeutic. This is demonstrated in the presentinvention by increased exon inclusion in the SMN2 gene in manycompartments of the brain and spinal cord, and also skeletal muscle. Invivo, the results described herein show an increase in exon inclusion ofup to 60% when using peptide conjugates of the invention compared withthe exon inclusion results of the same antisense oligonucleotidetherapeutic conjugated to a currently available peptide. This is asignificant improvement in the ability of such peptide carriers topenetrate cells of the central nervous system, which has historicallybeen a difficult area in which to target therapeutics to due to thepresence of the blood brain barrier. Increased cell penetration mayallow the present peptides to carry therapeutic molecules into areas ofthe brain and central nervous system where direct therapeutic action hasthus far not been available. This is of particular interest in thetreatment of genetic disorders such as SMA, DMD, Alzheimer's,Parkinson's, etc.

Without wishing to be bound by theory, the inventors believe that theinclusion of at least one glycosylated amino acid residue may allow thepeptide carriers to bind to sugar transporters (such as GLUT 1) locatedin the blood brain barrier. This transporter binding may assist thepeptides across the blood brain barrier, thereby facilitating improveddelivery of any conjugated therapeutic molecule directly into thecentral nervous system. Alternatively, it is thought that anotherproperty of the glycosylated residue, such as its effect in altering thehydrophobic/hydrophilic balance of the peptide, may result in enhancedblood brain barrier uptake by adsorptive transcytosis.

It was completely unanticipated that such a glycosylation modificationdirect to the amino acid sequence would boost the ability of anarginine-rich carrier cell-penetrating peptide to transport atherapeutic molecule cargo, such as an oligonucleotide, into both brainand spinal cord. Furthermore, it was unexpected that such transportwould be significantly enhanced so as to result in a therapeuticmolecule such as an antisense oligonucleotide successfully increasingexon inclusion in brain and spinal cord compartments of the centralnervous system as demonstrated herein.

For the avoidance of doubt, and in order to clarify the way in which thepresent disclosure is to be interpreted, certain terms used inaccordance with the present invention will now be defined further.

The invention includes any combination of the aspects and featuresdescribed except where such a combination is clearly impermissible orexpressly avoided.

The section headings used herein are for organisational purposes onlyand are not to be construed as limiting the subject matter described.

Amino Acid Nomenclature

References to ‘*’ throughout denote direct glycosylation of the relevantamino acid residue.

References to ‘X’ throughout denote the non-natural amino acid residueaminohexanoic acid.

References to ‘B’ throughout denote the non-natural amino acid residuebeta-alanine.

References to ‘Hyp’ throughout denote the amino acid residuehydroxyproline.

References to ‘Ac’ throughout denote acetylation of the relevant aminoacid residue.

References to ‘Cy’ throughout denote the non-natural amino acid1-(amino)cyclohexanecarboxylic acid.

References to ‘Az’ throughout denote the non-natural amino acid3-azetidine-carboxylic acid.

References to ‘Z’ throughout denote the non-natural amino acidtetrahydroisoquinoline-3-carboxylic acid (TIC).

References to ‘non-natural’ amino acid denote any amino acid that is notnaturally occurring and includes synthetic amino acids, modified aminoacids, spacers, and non-peptide bonded spacers. These non-natural aminoacids may also be referred to as amino acid analogues, and the term‘amino acid analogue’ is used interchangeably with the term ‘non-naturalamino acid’ throughout this specification. Suitable non-natural aminoacids that may be used in the present invention are: beta-alanine (B),aminohexanoic acid (X), tetrahydroisoquinoline-3-carboxylic acid (TIC),4-aminobutyryl (Aib), 1-(amino)cyclohexanecarboxylic acid (Cy), and3-azetidine-carboxylic acid (Az), for example.

References to other capital letters throughout denote the relevant aminoacid residue in accordance with the accepted alphabetic amino acid code.

Glycosylated Amino Acid Residue

The present invention defines a peptide comprising at least one directlyglycosylated amino acid residue.

By ‘directly glycosylated’ it is meant that the sugar is covalentlybonded to an atom in the amino acid residue.

Suitably the sugar is covalently bonded to an atom in the amino acidresidue without the presence of an intermediate spacer moiety.

By ‘spacer moiety’ we mean any chemical structure, for example: alkyl,alkenyl, alkynyl, aryl, ether, thioether, triazole, amide, carboxamide,urea, thiourea, semicarbazide, carbazide, hydrazine, oxime, phosphate,phosphoramidate, thiophosphate, boranophosphate or iminophosphate, andthe like, which may be placed between the amino acid residue and thesugar.

Suitably, the at least one directly glycosylated amino acid residue isO-linked glycosylated, N-linked glycosylated or S-linked glycosylated.

Suitably, the at least one directly glycosylated amino acid residue isglycosylated at any suitable functional group present in its side chain.

Suitably, the at least one directly glycosylated amino acid residue maybe glycosylated at one or more functional groups present in its sidechain.

Therefore, suitably the at least one directly glycosylated amino acidresidue may be glycosylated at multiple functional groups present in itsside chain.

Suitably, the at least one directly glycosylated amino acid residue isglycosylated at an OH, NH₂, NH₃ or SH functional group present in itsside chain.

Suitably the at least one directly glycosylated amino acid residue isselected from serine, cysteine, threonine, asparagine, glutamine,aminoproline, hydroxyproline, tyrosine, lysine, or amino acid analoguesthereof.

Suitably, the at least one directly glycosylated amino acid residue isglycosylated at an OH functional group present in its side chain.

Suitably, the at least one directly glycosylated amino acid residue isselected from a glycosylated serine, asparagine, threonine, or tyrosine,or amino acid analogues thereof.

In one embodiment, the at least one directly glycosylated amino acidresidue is glycosylated serine. In one embodiment, each of the directlyglycosylated amino acid residues is glycosylated serine.

In one embodiment, the at least one directly glycosylated amino acidresidue is glycosylated asparagine. In one embodiment, each of thedirectly glycosylated amino acid residues is glycosylated asparagine.

Suitably, the at least one directly glycosylated amino acid residue maybe selected from L or D enantiomers.

Suitably, the glycosylated serine may be selected from glycosylatedL-Serine, or glycosylated D-Serine.

Suitably, the glycosylated asparagine may be selected from glycosylatedL-Asparagine, or glycosylated D-Asparagine.

Suitably, the peptide comprises 1-3 directly glycosylated amino acidresidues. Suitably, the directly glycosylated amino acid residues may becontiguous with one another.

Suitably, the peptide may comprise directly glycosylated amino acidresidue sequences selected from the group consisting of: aa*, aa*aa*,and aa*aa*aa* (where “aa” represents an amino acid residue).

In one embodiment, the peptide comprises only 1 directly glycosylatedamino acid residue.

The at least one directly glycosylated amino acid residue may beglycosylated with any suitable sugar. Suitably the glycosylating sugarmay be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide,oligosaccharide or polysaccharide.

Suitable sugars for glycosylation are, for example; glucose, allose,altrose, idose, gulose, talose, xylose, mannose, galactose,mannoseamine, glucosamine, galactosamine, N-acetylgalactosamine,N-acetylglucosamine, 2-Acetylamino glucose, D-2-Acetylamino Glucose,lactose, maltose, isomaltose, or trehalose or sialic acid.

Suitably, the at least one directly glycosylated amino acid residue isglycosylated with glucose, 2-Acetylamino glucose, D-2-AcetylaminoGlucose, mannose, lactose, or galactose.

Suitably, the sugars used for glycosylation of the (or each)glycosylated amino acid residue may be modified or unmodified sugars.

The sugars used for glycosylation of the (or each) glycosylated aminoacid residue may be selected from L or D enantiomers, α or β anomers, orany other stereochemical variant.

In one embodiment, the sugar used for glycosylation of the (or each)glycosylated amino acid residue is a D enantiomer.

In one embodiment, the sugar used for glycosylation of the (or each)glycosylated amino acid residue is a β anomer.

In one embodiment, the at least one amino acid residue is directlyglycosylated with glucose. Therefore in one embodiment the at least oneamino acid residue is directly glucosylated.

In one embodiment, the at least one amino acid residue is directlyglucosylated with β-D glucose.

In one embodiment, the at least one amino acid residue is directlyglucosylated with D-2-Acetylamino Glucose

Suitably the sugar is covalently bonded to an atom of the at least oneamino acid residue. Suitably the sugar is covalently bonded to the aminoacid residue via an —O— linkage, —S— linkage or —N-linkage.

By the term ‘-O-linkage’ it is meant that the sugar is covalently bondedto an Oxygen atom present in the side chain of the at least one aminoacid residue.

By the term ‘—N-linkage’ it is meant that the sugar is covalently bondedto a Nitrogen atom present in the side chain of the at least one aminoacid residue.

By the term ‘-S-linkage’ it is meant that the sugar is covalently bondedto a Sulphur atom present in the side chain of the at least one aminoacid residue.

In one embodiment, the sugar is covalently bonded to the amino acidresidue by via an —O— linkage.

In one embodiment, the sugar is glucose and is covalently bonded to theat least one amino acid residue by and —O— linkage. In one embodiment,the at least one directly glycosylated amino acid residue isβ-D-glucosyl serine.

A β-D-glucosyl serine has the following chemical structure:

In one embodiment, the glycosylated amino acid residue is L-serineglycosylated with a D-Glucose sugar.

In one embodiment, the glycosylated amino acid residue is D-serineglycosylated with a D-Glucose sugar.

In one embodiment, the at least one directly glycosylated amino acidresidue is β-D-2-Acetylamino glucosyl asparagine.

In one embodiment, the glycosylated amino acid residue is L-asparagineglycosylated with a D-2-Acetylamino Glucose sugar.

In one embodiment, the glycosylated amino acid residue is D-asparagineglycosylated with a D-2-Acetylamino Glucose sugar.

In one embodiment, the peptide comprises one directly glycosylatedserine residue.

In one embodiment, the peptide comprises one directly glycosylatedasparagine residue.

Optionally, the at least one directly glycosylated amino acid residuemay be present with, and contiguous with, one or more hydrophobic coredomains as defined below.

Suitably, the at least one directly glycosylated amino acid residue iscontiguous with one hydrophobic core domain

Alternatively, the at least one directly glycosylated amino acid residuemay be present without a contiguous hydrophobic core domain.

In a suitable embodiment the at least one directly glycosylated aminoacid residue is contiguous with an arginine-rich arm domain.

Suitably, the at least one directly glycosylated amino acid residue,optionally together with one or more hydrophobic core domains, isflanked on both sides by arginine-rich arm domains. Therefore, suitably,the peptide may comprise the following structures:

-   -   [arginine-rich arm domain]-[aa*]-[hydrophobic core        domain]-[arginine-rich arm domain]    -   [arginine-rich arm domain]-[hydrophobic core        domain]-[aa*]-[arginine-rich arm domain]

Alternatively, or additionally, the peptide may comprise the followingstructure:

-   -   [arginine-rich arm domain]-[aa*]-[arginine-rich arm domain]

Alternatively, the at least one directly glycosylated amino acidresidue, optionally together with one or more hydrophobic core domains,is flanked on one side by an arginine-rich arm domain.

Therefore, suitably, the peptide may comprise the following structures:

-   -   [aa*]-[hydrophobic core domain]-[arginine-rich arm domain]    -   [hydrophobic core domain]-[aa*]-[arginine-rich arm domain]    -   [arginine-rich arm domain]-[aa*]-[hydrophobic core domain]    -   [arginine-rich arm domain]-[hydrophobic core domain]-[aa*]

Alternatively, or additionally, the peptide may comprise the followingstructure:

-   -   [aa*]-[arginine-rich arm domain]    -   [arginine-rich arm domain]-[aa*]

Suitably, the peptide may comprise more than one of the above-describedstructures and in any combination. Further peptide structures within thescope of the present invention are described below.

Arm Domains

The present invention defines a peptide comprising one or morearginine-rich arm domains.

An ‘arginine-rich’ arm domain may comprise at least 30% arginineresidues. For example, an arginine-rich arm domain may comprise at least35%, at least 40%, at least 45%, or at least 50% arginine residues.Suitably an arginine-rich arm domain may comprise at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95%, or more, arginine residues. In asuitable embodiment an arginine-rich arm domain may comprise 100%arginine residues.

An arginine-rich arm domain may comprise more Arginine residues than anyother single amino acid residue.

Suitably the arginine-rich arm domains comprise a combined total ofbetween 5-10 Arginine residues, suitably between 8-10 Arginine residues.Thus it will be appreciated that the arginine-rich arm domains presentin a peptide of the invention may comprise a combined total of 5arginine residues, a combined total of 6 arginine residues, a combinedtotal of 7 arginine residues, a combined total of 8 arginine residues, acombined total of 9 arginine residues, or a combined total of 10arginine residues.

Suitably the arginine-rich arm domains comprise no more than 3contiguous Arginine residues, suitably no more than 2 contiguousArginine residues.

Suitably, each arginine-rich arm domain comprises a length of between1-12 amino acid residues, suitably a length of between 6-9 amino acidresidues.

Suitably the arginine-rich arm domains are cationic.

Suitably, the arginine-rich arm domains comprise amino acid residuesselected from the group consisting of: arginine, alanine, beta-alanine,histidine, proline, glycine, cysteine, tryptophan, hydroxyproline,aminohexanoic acid, 3-azetidine-carboxylic acid (Az), and1-(amino)cyclohexanecarboxylic acid (Cy), and amino acid analoguesthereof, or any other non-natural amino acid. Suitably, thearginine-rich arm domains comprise amino acid residues selected from thegroup consisting of: arginine, beta-alanine and aminohexanoic acid.

Suitably, the arginine-rich arm domains consist of amino acid residuesselected from the group consisting of: arginine, alanine, beta-alanine,histidine, proline, glycine, cysteine, tryptophan, hydroxyproline,aminohexanoic acid, 3-azetidine-carboxylic acid (Az), and1-(amino)cyclohexanecarboxylic acid (Cy).

Suitably, the arginine-rich arm domains consist of amino acid residuesselected from the group consisting of: arginine, beta-alanine andaminohexanoic acid.

The use of non-natural amino acid residues such as beta-alanine oraminohexanoic acid is advantageous in that it helps minimise theimmunogenicity of the peptide and also helps to improve thebiostability, particularly to metabolism by protease enzymes.

Suitable non-natural amino acids that may be used in the arginine richarm domains of the present invention are: beta-alanine (B),aminohexanoic acid (X), 3-azetidine-carboxylic acid (Az), and1-(amino)cyclohexanecarboxylic acid (Cy).

Suitably, the arginine-rich arm domains are formed of amino acid unitsselected from: R, RR, RJR, RRJ, JRR in any combination or order. WhereinJ represents any non-natural amino acid.

Suitably, the arginine-rich arm domains are formed of amino acid unitsselected from: R, RR, RBR, RXR, XXR, XRR, RRX, BXR, RXB, XRB, RBB, BRB,BBR, RRB, BRR, and BRX in any combination or order. Suitably, anarginine-rich arm domain may consist of one of these units, or amultiple of these units.

Suitably, the arginine-rich arm domains are formed of amino acid unitsselected from: RRRRRRR (SEQ ID NO.136), RJRRRRR (SEQ ID NO.137), RRJRRRR(SEQ ID NO.138), RRRJRRR (SEQ ID NO.139), RRRRJRR (SEQ ID NO.140),RRRRRJR (SEQ ID NO.141), RJRRJ[RRR]n (SEQ ID NOs.142, 176, 177, 178,179), RRJRRJ[RR]n (SEQ ID NOs.143, 180, 181, 182, 183), RRRJRRJR (SEQ IDNOs.144), RJRRRJ[RR]n (SEQ ID NOs.145, 184, 185, 186, 187), RJ[RRRR]nJR(SEQ ID NOs.146, 188, 189, 190, 191);

Wherein J represents any non-natural amino acid and wherein n representsan integer from 1-5.

Suitably, wherein n represents an integer from 1-3, suitably wherein nrepresents, 1, 2 or 3.

Suitably, each arginine-rich arm domain is selected from one of thefollowing sequences: RXRRBRRXR (SEQ ID NO.81), RXRBRXR (SEQ ID NO.82),RXRRBRR (SEQ ID NO.83), RBRXR (SEQ ID NO.84), RBRRBRRBR (SEQ ID NO.85),RBRBRBR (SEQ ID NO.86), RGRRGRRGR (SEQ ID NO.87), RGRGRGR (SEQ IDNO.88), RPRRPRRPR (SEQ ID NO.89), RPRPRPR (SEQ ID NO.90),RHypRRHypRRHypR (SEQ ID NO.91), RHypRHypRHypR (SEQ ID NO.92), RARRARRAR(SEQ ID NO.93), RARARAR (SEQ ID NO.94), RCy*RRCy*RRCy*R (SEQ ID NO.95),RCy*RCy*RCy*R (SEQ ID NO.96), RRBRRBR (SEQ ID NO.97), RBRRBR (SEQ IDNO.98, RRBR (SEQ ID NO.99), RBR, R, RBRBR (SEQ ID NO.100), RBRBRR (SEQID NO.101), RBRRR (SEQ ID NO.102), RRRR (SEQ ID NO.103), RBRRBRRR (SEQID NO.104), RBRRRRR (SEQ ID NO.105), RRRRRR (SEQ ID NO.106), RRBRR (SEQID NO.107), RGRR (SEQ ID NO.108), GRRGR (SEQ ID NO.109), RGGRBRGGR (SEQID NO.110), RXRRBRRXRRXRBRXR (SEQ ID NO.113), RXRR (SEQ ID NO.114, RRXR(SEQ ID NO.115), RXR, RRBRBRXR (SEQ ID NO.117), RRBRRBRBRXR (SEQ IDNO.118), RXRRBRRBR (SEQ ID NO.119), RXRRBRRBRBR (SEQ ID NO.120), RXRRBR(SEQ ID NO.121), RXRBRR (SEQ ID NO.122), HXHRBRRXR (SEQ ID NO.123),RXHBHXR (SEQ ID NO.124), RR, RXRXR (SEQ ID NO.125), BRBRBR (SEQ IDNO.127), BRKBRKRBBR (SEQ ID NO.128), BRKBRKRBBRK (SEQ ID NO.129),RAzRRAzRR (SEQ ID NO.130), RAzRAzR (SEQ ID NO.131), and RXRBR (SEQ IDNO.132).

Suitably, each arginine-rich arm domain is selected from one of thefollowing sequences: RXRRBRRXR (SEQ ID NO.81), RXRBRXR (SEQ ID NO.82),RXRRBRR (SEQ ID NO.83), RBRXR (SEQ ID NO.84), RBRBR (SEQ ID NO.100),RBRRBR (SEQ ID NO.98), RXRBR (SEQ ID NO.132), and RXRXR (SEQ ID NO.125).

Optionally, the amino acid residues of the arginine-rich arm domains mayalso be glycosylated. Details regarding glycosylation may be found inthe section describing the glycosylated amino acid residue.

Suitably, each of the arginine-rich arm domains comprised in the peptideis of similar length. For example, the length of each arginine-rich armdomain may differ from one another by no more than 1, 2, or 3 amino acidresidues. In a suitable embodiment each arginine-rich arm domain presentin a peptide of the invention may be of equal length to one another.

Suitably, each of the arginine-rich arm domains comprised in the peptidehas the same sequence.

Suitably, the peptide comprises up to 4 arginine-rich arm domains, 3arginine-rich arm domains, or 2 arginine-rich arm domains.

In one embodiment, the peptide comprises 2 arginine-rich arm domains.

Suitably, each of the arginine-rich arm domains comprised in the peptideis separated by a directly glycosylated amino acid residue, optionallytogether with a hydrophobic core domain.

In one embodiment, the peptide comprises two arginine-rich arm domainsflanking one central directly glycosylated amino acid residue.

In one embodiment, the peptide comprises two arginine-rich arm domainsflanking one central directly glycosylated amino acid residue contiguouswith a hydrophobic core domain.

Suitably, the peptide comprises a first arginine-rich arm domainselected from the following sequences: RXRRBRRXR (SEQ ID NO.81), RXRRBRR(SEQ ID NO.83), RBRBR (SEQ ID NO.100), and RXRXR (SEQ ID NO.125).

Suitably, the peptide comprises a second arginine-rich arm domainselected from the following sequences: RXRBRXR (SEQ ID NO.82), RBRXR(SEQ ID NO.84), RBRRBR (SEQ ID NO.98), RXRBR (SEQ ID NO.132), RBRBR (SEQID NO.100), and RXRXR (SEQ ID NO.125).

In one embodiment, the peptide comprises a first arginine-rich armdomain consisting of the sequence RXRRBRRXR (SEQ ID NO.81) and a secondarginine-rich arm domain consisting of the sequence RXRBRXR (SEQ IDNO.82).

In one embodiment, the peptide comprises a first arginine-rich armdomain consisting of the sequence RXRRBRR (SEQ ID NO.83) and a secondarginine-rich arm domain consisting of the sequence RBRXR (SEQ IDNO.84).

In one embodiment, the peptide comprises a first arginine-rich armdomain consisting of the sequence RXRRBRR (SEQ ID NO.83) and a secondarginine-rich arm domain consisting of the sequence RXRBR (SEQ IDNO.132).

In one embodiment, the peptide comprises a first arginine-rich armdomain consisting of the sequence RBRBR (SEQ ID NO.100) and a secondarginine-rich arm domain consisting of the sequence RBRBR (SEQ IDNO.100).

In one embodiment, the peptide comprises a first arginine-rich armdomain consisting of the sequence RXRXR (SEQ ID NO.125) and a secondarginine-rich arm domain consisting of the sequence RXRXR (SEQ IDNO.125).

Hydrophobic Core Domains

The present invention defines a peptide comprising at least one directlyglycosylated amino acid residue and one or more arginine-rich armdomains.

The peptide may further comprise one or more hydrophobic core domains.

By ‘hydrophobic’ it is meant that a core domain, when taken as a whole,has an overall hydrophobic nature. This may be determined by, forexample, hydropathy plot, such as a Kyte-Doolittle hydropathy plot (inwhich case the hydrophobicity of the core domain will be indicated by anoverall positive score). Suitably, a core domain may comprise a majorityof hydrophobic amino acid residues.

Suitably the hydrophobic core domains each comprise between 1-4hydrophobic amino acid residues.

In one embodiment, the hydrophobic core domains each comprise between1-2 hydrophobic amino acid residues.

Suitable hydrophobic amino acid residues that may be incorporated in thecore domains are, for example; glycine, alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, and tryptophan, or aminoacid analogues thereof.

Suitably the hydrophobic amino acid residues of the core domains areselected from phenylalanine or leucine.

Suitably each hydrophobic core domain comprises a total of between 1-6amino acid residues, suitably 1-5 amino acid residues, suitably 1-4amino acid residues, suitably 1-3 amino acid residues.

In one embodiment, each hydrophobic core domain comprises a total ofbetween 1-2 amino acid residues.

The other amino acid residues present in each hydrophobic core domain inaddition to the hydrophobic amino acid residues may be any other aminoacid, for example; arginine, asparagine, aspartic acid, cysteine,glutamic acid, glutamine, glycine, histidine, lysine, serine, threonine,tyrosine, or any amino acid analogues thereof.

Suitably, the other amino acids residues present in each core domain areselected from tyrosine and glutamine.

Optionally the hydrophobic core domain may include one or morenon-natural amino acids such as aminohexanoic acid or β-Alanine ortetrahydroisoquinoline-3-carboxylic acid (TIC) as listed above.

Suitably, the hydrophobic core domain may comprise one of the followingsequences: ZAA, ZA, Z, AZA, AZ, ZAZ, ZZA and ZZZ;

Wherein Z represents 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(TIC) residue.

Wherein A represents a hydrophobic amino acid residue as defined above.

Suitably the peptide comprises up to 4 hydrophobic core domains.Suitably the peptide comprises 3 hydrophobic core domains, suitably 2hydrophobic core domains, or suitably only a single hydrophobic coredomain.

In one embodiment, the peptide comprises only 1 hydrophobic core domain.

Suitably the hydrophobic core domains are contiguous with the at leastone directly glycosylated amino acid residue. Suitably the hydrophobiccore domains may be on the C-terminal or N-terminal side of the at leastone directly glycosylated amino acid residue.

In one embodiment, the hydrophobic core domains are on the C-terminalside of the at least one directly glycosylated amino acid residue.

Suitably the hydrophobic core domains contiguous with at least onedirectly glycosylated amino acid residue may be positioned anywherewithin the peptide.

In a suitable embodiment a hydrophobic core domain contiguous with atleast one directly glycosylated amino acid residue is positioned in thepeptide such that the directly glycosylated amino acid residue iscontiguous with an arginine-rich arm domain.

Suitably, the hydrophobic core domains contiguous with the directlyglycosylated amino acid residue are positioned between two flanking armdomains.

Suitably at least one of the hydrophobic core domains contiguous with adirectly glycosylated amino acid residue is positioned at the centre ofthe peptide.

In one embodiment, the peptide comprises one hydrophobic core domaincontiguous with a directly glycosylated amino acid residue positioned atthe centre of the peptide between two flanking arm domains.

Suitably the hydrophobic core domains are selected from one of thefollowing sequences: GFTGPL (SEQ ID NO.133), QFL, Z, ZL, F, FL, FQILY(SEQ ID NO.134), FQ, WF, QF, FQ, and YQFLI (SEQ ID NO.135). Suitably,the core domains are selected from one of the following sequences Z, Fand FL.

In one embodiment, the peptide comprises one core domain wherein thecore domain consists of the sequence F.

In one embodiment, the peptide comprises one core domain wherein thecore domain consists of the sequence FL.

In one embodiment, the peptide comprises one core domain wherein thecore domain consists of the sequence Z.

Peptide Structure

The present invention defines a peptide comprising at least one directlyglycosylated amino acid residue and one or more arginine-rich armdomains, optionally in combination with at least one hydrophobic coredomain.

Suitably, the peptides have a sequence that is a contiguous singlemolecule. Suitably, the peptide comprises several domains and at leastone directly glycosylated amino acid residue in a linear arrangementbetween the N-terminus and the C-terminus. Suitably, the domains areselected from arginine rich arm domains and optional hydrophobic coredomains described above.

Suitably, the peptide sequence is comprised of amino acid residues andoptional non-natural amino acid residues, or amino acid analogues asdefined hereinabove. Accordingly in some case, the peptide may comprisenon-peptide bonds. The relevant amino acid residues comprised in the atleast one directly glycosylated residue and for the domains of thepeptide are described in each section above.

Each domain and the at least one directly glycosylated amino acidresidue have common sequence characteristics as described in thesections above, but the exact sequence of each domain is capable ofvariation and modification. Thus a range of sequences is possible foreach domain. The combination of each possible domain sequence and theoptions for the or each directly glycosylated amino acid residue yieldsa range of peptide structures, each of which form part of the presentinvention. Features of the peptide structures are described below.

Suitably, the directly glycosylated amino acid residues separate eacharginine-rich arm domain. Suitably, each directly glycosylated aminoacid residue is flanked by arginine-rich arm domains on either sidethereof.

Suitably no arginine-rich arm domain is contiguous with anotherarginine-rich arm domain.

In one embodiment, the peptide comprises three directly glycosylatedamino acid residues separated by arginine-rich arm domains and flankedon either side by two further arginine-rich arm domains in the followingarrangement:

-   -   [arginine-rich arm domain]-[aa*]-[arginine-rich arm        domain]-[aa*]-[arginine-rich arm domain]-[aa*]-[arginine-rich        arm domain]

In one embodiment, the peptide comprises two directly glycosylated aminoacid residues separated by one arginine-rich arm domain and flanked oneither side by two further arginine-rich arm domains in the followingarrangement:

-   -   [arginine-rich arm domain]-[aa*]-[arginine-rich arm        domain]-[aa*]-[arginine-rich arm domain]

In one embodiment, the peptide comprises one directly glycosylated aminoacid residue flanked by two arginine-rich arm domains in the followingarrangement:

-   -   [arginine-rich arm domain]-[aa*]-[arginine-rich arm domain]

Optionally, more than one contiguous directly glycosylated amino acidresidue may be present in the peptide.

Therefore, in one embodiment, the peptide may comprise three contiguousdirectly glycosylated amino acid residues flanked by two arginine-richarm domains in the following arrangement:

-   -   [arginine-rich arm domain]-[aa*]-[aa*]-[aa*]-[arginine-rich arm        domain]

Therefore, in one embodiment, the peptide may comprise two contiguousdirectly glycosylated amino acid residues flanked by two arginine-richarm domains in the following arrangement:

-   -   [arginine-rich arm domain]-[aa*]-[aa*]-[arginine-rich arm        domain]

Optionally, the directly glycosylated amino acid residues may be presentin combination with a hydrophobic core domain. If present, suitably thedirectly glycosylated amino acid residues are contiguous with thehydrophobic core domain. The hydrophobic core domain may be present onthe C-terminal or the N-terminal side of the directly glycosylated aminoacid residue.

Therefore in one embodiment, the peptide may comprise one directlyglycosylated amino acid residue contiguous with one hydrophobic coredomain flanked by two arginine-rich arm domains in the followingarrangement:

-   -   [arginine-rich arm domain]-[aa*]-[hydrophobic core        domain]-[arginine-rich arm domain]

In one embodiment, the peptide may comprise two directly glycosylatedamino acid residues each contiguous with one hydrophobic core domainseparated by one arginine-rich arm domain and flanked on either side bytwo further arginine-rich arm domains in the following arrangement:

-   -   [arginine-rich arm domain]-[aa*]-[hydrophobic core        domain]-[arginine-rich arm domain]-[aa*]-[hydrophobic core        domain]-[arginine-rich arm domain]

In one embodiment, the peptide may comprise three directly glycosylatedamino acid residues each contiguous with one hydrophobic core domainseparated by arginine-rich arm domains and flanked on either side by twofurther arginine-rich arm domains in the following arrangement:

-   -   [arginine-rich arm domain]-[aa*]-[hydrophobic core        domain]-[arginine-rich arm domain]-[aa*]-[hydrophobic core        domain]-[arginine-rich arm domain]-[aa*]-[hydrophobic core        domain]-[arginine-rich arm domain]

Suitably, any combination of the above peptide structures is envisagedby the present invention. Each directly glycosylated amino acid residuemay be present with or without a contiguous hydrophobic core domain.Multiple contiguous directly glycosylated amino acid residues may bepresent.

Suitably, the peptide may comprise a first directly glycosylated aminoacid residue without a contiguous hydrophobic core domain and a seconddirectly glycosylated amino acid residue contiguous with a hydrophobiccore domain, for example.

Therefore, in one embodiment, the peptide may comprise two directlyglycosylated amino acid residues, the first without a hydrophobic coredomain and the second contiguous with a hydrophobic core domain, eachseparated by an arginine-rich arm domain and flanked on either side bytwo further arginine-rich arm domains in the following arrangement:

-   -   [arginine-rich arm domain]-[aa*]-[arginine-rich arm        domain]-[aa*]-[hydrophobic core domain]-[arginine-rich arm        domain]

Therefore, in one embodiment, the peptide may comprise two directlyglycosylated amino acid residues, the first contiguous with ahydrophobic core domain and the second without a hydrophobic coredomain, each separated by an arginine-rich arm domain and flanked oneither side by two further arginine-rich arm domains in the followingarrangement:

-   -   [arginine-rich arm domain]-[aa*]-[hydrophobic core        domain]-[arginine-rich arm domain]-[aa*]-[arginine-rich arm        domain]

Therefore, in one embodiment, the peptide may comprise three directlyglycosylated amino acid residues, the first and third without ahydrophobic core domain, and the second contiguous with a hydrophobiccore domain, each separated by an arginine-rich arm domain and flankedon either side by two further arginine-rich arm domains in the followingarrangement:

-   -   [arginine-rich arm domain]-[aa*]-[arginine-rich arm        domain]-[aa*]-[hydrophobic core domain]-[arginine-rich arm        domain]-[aa*]-[arginine-rich arm domain]

Suitably, the peptide is N-terminal modified.

Suitably the peptide is N-acetylated, N-methylated,N-trifluoroacetylated, N-trifluoromethylsulfonylated, orN-methylsulfonylated.

Optionally, the N-terminus of the peptide may be unmodified.

Suitably, the peptide is C-terminal modified.

Suitably, the peptide comprises a C-terminal modification selected from:Carboxy-, Thioacide-, Aminooxy-, Hydrazino-, thioester-, azide, strainedalkyne, strained alkene, aldehyde-, thiol or haloacetyl-group.

Advantageously, the C-terminal modification provides a means for linkageof the peptide to the therapeutic molecule.

Accordingly, the C-terminal modification may comprise the linker andvice versa. Suitably, the C-terminal modification may consist of thelinker or vice versa. Suitable linkers are described herein elsewhere.

Suitably, the peptide comprises a C-terminal carboxyl group.

Suitably, the C-terminal carboxyl group may be a provided by a glycine,aminohexanoic acid, β-alanine, alanine, glutamic acid side chain oraspartic acid side chain.

Suitably, the C-terminal carboxyl group is provided by a glycine orβ-alanine residue.

In one embodiment, the C terminal carboxyl group is provided by aglycine residue.

In one embodiment, the C terminal carboxyl group is provided by aβ-alanine residue.

In one embodiment, the C-terminal modification comprises a glycineresidue and/or a β-alanine residue.

In one embodiment, the C-terminal modification is a glycine residue.

In one embodiment, the C-terminal modification is a β-alanine residue.

Alternatively, the peptide may be linked to the therapeutic moleculethrough an N-terminal modification of the peptide.

Suitably, in such embodiments, the peptide comprises an N-terminalmodification selected from: succinic acid, a side-chain of aspartic acidor a side chain of glutamic acid.

Suitably, in such embodiments, the C-terminus of the peptide is presentas an amide.

The peptide of the present invention is defined as having a total lengthof 40 amino acid residues or less. The peptide may therefore be regardedas an oligopeptide.

Suitably, the peptide comprises a total length of between 3-30 aminoacid residues, suitably of between 5-25 amino acid residues, of between10-25 amino acid residues, of between 13-23 amino acid residues, ofbetween 15-21 amino acid residues.

Suitably the peptide is capable of penetrating cells. The peptide maytherefore be regarded as a cell penetrating peptide.

Suitably, the peptide is for attachment to a therapeutic molecule.Suitably, the peptide is for transporting a therapeutic molecule into atarget cell. Suitably, the peptide is for delivering a therapeuticmolecule into a target cell. The peptide may therefore be regarded as acarrier peptide.

Suitably, the peptide may be selected from any of the followingsequences:

(SEQ ID NO. 8) RXRRBRRXRQFLRXRBRXRS* (SEQ ID NO. 9) RXRRBRRXRQFLRXRS*RXR(SEQ ID NO. 10) RXRRS*RRXRQFLRXRBRXR (SEQ ID NO. 11)S*RXRRBRRXR QFL RXRBRXR (SEQ ID NO. 12) RXRRBRRXR S*QFLS*RXRBRXR(SEQ ID NO. 13) RXRRBRRXR S*QFLRXRBRXR (SEQ ID NO. 14)RXRRBRRXRS*FLRXRBRXR (SEQ ID NO. 15) (S*BRKBRKRBBR)₂K (SEQ ID NO. 16)(GFTGPLS*BRKBRKRBBR)₂K (SEQ ID NO. 17) RXRRBRRFS*RBRXR (SEQ ID NO. 18)RBRRBRRBRS*FLRBRBRBR (SEQ ID NO. 19) RGRRGRRGRS*FLRGRGRGR(SEQ ID NO. 20) RPRRPRRPRS*FLRPRPRPR (SEQ ID NO. 21)RHypRRHypRRHypRS*FLRHypRHypRHypR (SEQ ID NO. 22) RARRARRARS*FLRARARAR(SEQ ID NO. 23) RCyRRCyRRCyRS*FLRCyRCyRCyR (SEQ ID NO. 24)RRBRRBRS*FLRBRBRBR (SEQ ID NO. 25) RBRRBRS*FLRBRBRBR (SEQ ID NO. 26)RRBRS*FLRBRBRBR (SEQ ID NO. 27) RBRS*FLRBRBRBR (SEQ ID NO. 28)RS*FLRBRBRBR (SEQ ID NO. 29) RBRRBRRBRS*FLRBRBR (SEQ ID NO. 30)RBRRBRRBRS*FLRBR (SEQ ID NO. 31) RBRRBRRBRS*FLR (SEQ ID NO. 32)RBRRBRRBRS*FL (SEQ ID NO. 33) RBRRBRRBRS*FLRBRBRR (SEQ ID NO. 34)RBRRBRRBRS*FLRBRRR (SEQ ID NO. 35) RBRRBRRBRS*FLRRRR (SEQ ID NO. 36)RBRRBRRRS*FLRBRBRBR (SEQ ID NO. 37) RBRRRRRS*FLRBRBRBR (SEQ ID NO. 38)RRRRRRS*FLRBRBRBR (SEQ ID NO. 39) RBRRBRRRS*FLRRBRR (SEQ ID NO. 40)RBRRRRRS*FLRRRR (SEQ ID NO. 41) RRRRRRS*FLRRRR (SEQ ID NO. 42)RGRR S*GRRGRS*FLRGGRBRGGR (SEQ ID NO. 43) RXRRBRRXRS*FRXRBRXR(SEQ ID NO. 44) RXRRBRRXRS*RXRBRXR (SEQ ID NO. 45) RXRRBRRS*FQILYRBRXR(SEQ ID NO. 46) RXRRBRRS*FLRBRXR (SEQ ID NO. 47)RXRRBRRXRS*FLRXRBRXRS*FL (SEQ ID NO. 48) RXRRBRRXRRXRBRXRS*FL(SEQ ID NO. 49) RXRRS*RRXRS*FLRXRS*RXR (SEQ ID NO. 50)RXRRBRRXRS*FQRXRBRXR (SEQ ID NO. 52) RXRRBRRXRS*WFRXRBRXR(SEQ ID NO. 53) RXRRBRRXRS*QFRXRBRXR (SEQ ID NO. 54)RXRRBRRXRS*FQRXRBS*YQFLIRXR (SEQ ID NO. 55) RXRRBRRS*RBRXR(SEQ ID NO. 56) RXRRFS*RRBRBRXR (SEQ ID NO. 57) R FS*RRBRRBRBRXR(SEQ ID NO. 58) RXRRS*RRBRBRXR (SEQ ID NO. 59) RS*RRBRRBRBRXR(SEQ ID NO. 60) RXRRBRRBRS*RXR (SEQ ID NO. 61) RXRRBRRBRBRS*R(SEQ ID NO. 62) RXRRBRFS*RBR (SEQ ID NO. 63) RXRRBRS*RBR (SEQ ID NO. 64)RXRBRRS*RBR (SEQ ID NO. 65) RRBRRS*RBR (SEQ ID NO. 66)HXHRBRRXRS*RXHBHXR (SEQ ID NO. 67) RXRRBRRS*S*RBRXR (SEQ ID NO. 68)RXRRBRRS*S*S*RBRXR (SEQ ID NO. 69) RXRRS*RRS*RS*RXR (SEQ ID NO. 70)RBRBRS*RBRBR (SEQ ID NO. 71) RXRXRS*RXRXR (SEQ ID NO. 72) RXRRBS*BRBRBR(SEQ ID NO. 73) RXRRBRRZS* RBRXR (SEQ ID NO. 74) RXRRBRRFS¹*RBRXR(SEQ ID NO. 75) RXRRBRRFS²* RBRXR (SEQ ID NO. 76) RXRRBRRFS³* RBRXR(SEQ ID NO. 77) RXRRBRRFS⁴* RBRXR (SEQ ID NO. 78) RXRRBRRFN* RBRXR(SEQ ID NO. 79) RXRRBRRFS⁶* RBRXR (SEQ ID NO. 80) RAzRRAzRRZS*RAzRAzR

Suitably the peptide is selected from the following sequences:RXRRBRRXRS*FLRXRBRXR (SEQ ID NO.14), RXRRBRRFS*RBRXR (SEQ ID NO.17),RXRRBRRZS*RBRXR (SEQ ID NO.73), RXRRBRRFS¹*RBRXR (SEQ ID NO.74),RBRBRS*RBRBR (SEQ ID NO.70) and RXRXRS*RXRXR (SEQ ID NO.71).

Wherein Z represents 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.

Wherein S* represents L-serine glycosylated with D-Glucose sugar.

Wherein S¹* represents D-serine glycosylated with D-Glucose sugar.

Wherein S²* represents L-serine glycosylated with an L-Glucose sugar.

Wherein S³* represents L-serine glycosylated with a D-Mannose sugar.

Wherein S⁴* represents L-serine glycosylated with a D-Lactose sugar.

Wherein N* represents L-asparagine glycosylated with a D-2-AcetylaminoGlucose sugar.

Wherein S⁶* represents L-serine glycosylated with a Galactose sugar.

In one embodiment, the peptide is RXRRBRRXRS*FLRXRBRXR (SEQ ID NO.14).

In one embodiment, the peptide is RXRRBRRFS*RBRXR (SEQ ID NO.17).

In one embodiment, the peptide is RXRRBRRZS*RBRXR (SEQ ID NO.73).

In one embodiment, the peptide is RXRRBRRFS¹*RBRXR (SEQ ID NO.74).

In one embodiment, the peptide is RBRBRS*RBRBR (SEQ ID NO.70).

In one embodiment, the peptide is RXRXRS*RXRXR (SEQ ID NO.71).

Suitably the peptide is capable of penetrating into cells and tissues,suitably into the nucleus of cells. Suitably into muscle tissues.

Suitably the peptide is capable of penetrating into cells of the centralnervous system.

Suitably the peptide is capable of crossing the blood brain barrier,suitably into many compartments of the central nervous system.

Suitably the peptide is capable of crossing the blood brain barrier,suitably into the cortex, brainstem, cerebellum, cervical, thoracic, andlumber compartments of the central nervous system.

Optionally, the peptide may further be selected from any of thefollowing sequences:

(SEQ ID NO. 116) RXRRBRRZS* RXRBR (SEQ ID NO. 51) RXRRBRRFS¹* RXRBR(SEQ ID NO. 111) RXRRBRRFS²* RXRBR (SEQ ID NO. 112) RXRRBRRFS³* RXRBR(SEQ ID NO. 157) RXRRBRRFS⁴* RXRBR (SEQ ID NO. 126) RXRRBRRFN* RXRBR(SEQ ID NO. 158) RXRRBRRFS⁶* RXRBR (SEQ ID NO. 159) RXRRBRRFS⁵* RBRXR(SEQ ID NO. 160) RXRRBRRF S⁵* RXRBR (SEQ ID NO. 161) RBRBRS*RBRRBR(SEQ ID NO. 162) RXRRBRRS¹*RBRXR (SEQ ID NO. 163) RXRRBRRS²* RBRXR(SEQ ID NO. 164) RXRRBRRS³* RBRXR (SEQ ID NO. 165) RXRRBRRS⁴* RBRXR(SEQ ID NO. 166) RXRRBRRN* RBRXR (SEQ ID NO. 167) RXRRBRRS⁶* RBRXR(SEQ ID NO. 168) RXRRBRRS¹* RXRBR (SEQ ID NO. 169) RXRRBRRS²* RXRBR(SEQ ID NO. 170) RXRRBRRS³* RXRBR (SEQ ID NO. 171) RXRRBRRS⁴* RXRBR(SEQ ID NO. 172) RXRRBRRN* RXRBR (SEQ ID NO. 173) RXRRBRRS⁶* RXRBR(SEQ ID NO. 174) RXRRBRRS⁵* RBRXR (SEQ ID NO. 175) RXRRBRRS⁵* RXRBRWherein S⁵* represents L-serine glycosylatedwith a D-2-Acetylamino Glucose sugar.

Conjugates

The peptide of the invention may be covalently linked to a therapeuticmolecule in order to provide a conjugate.

The therapeutic molecule may be any molecule for treatment of a disease,suitably any small molecule. The therapeutic molecule may be selectedfrom: a nucleic acid, peptide nucleic acid, antisense oligonucleotide(such as PNA, PMO), short interfering RNA, micro RNA, peptide, cyclicpeptide, protein, pharmaceutical or drug.

In one embodiment, the therapeutic molecule is an antisenseoligonucleotide.

Suitably the antisense oligonucleotide is comprised of aphosphorodiamidate morpholino oligonucleotide (PMO).

Alternatively the oligonucleotide may be a modified PMO or any othercharge-neutral oligonucleotide such as a peptide nucleic acid (PNA), achemically modified PNA such as a gamma-PNA (Bahal, Nat. Comm. 2016),oligonucleotide phosphoramidate (where the non-bridging oxygen of thephosphate is substituted by an amine or alkylamine such as thosedescribed in WO2016028187A1, or any other partially or fullycharge-neutralized oligonucleotide.

The therapeutic antisense oligonucleotide sequence may be selected fromany that are available, for example antisense oligonucleotides for exonskipping in DMD are described in Yin, Mol. Ther. 2011; and Betts, MTNA,2012).

In one embodiment, the therapeutic antisense oligonucleotide iscomplementary to the ISSN1 or IN7 sequence, suitable antisenseoligonucleotides are described in Zhou, H G T, 2013; and Hammond et al,2016; and Osman et al, HMG, 2014.

PMO oligonucleotides of any sequence may be purchased (for example fromGene Tools Inc, USA)

In one embodiment, the therapeutic molecule of the conjugate is anoligonucleotide complementary to the pre-mRNA of a gene target.

Suitably, the oligonucleotide complementary to the pre-mRNA of a genetarget gives rise to a steric blocking event that alters the pre-mRNAleading to an altered mRNA and hence a protein of altered sequence.

Suitably the steric blocking event may be exon inclusion (or exonskipping).

Optionally, lysine residues may be added to one or both ends of atherapeutic molecule (such as a PMO or PNA) before attachment to thepeptide to improve water solubility.

Suitably the therapeutic molecule has a molecular weight of less than5,000 Da, suitably less than 3000 Da or suitably less than 1000 Da.

Suitably, the peptide is covalently linked to the therapeutic moleculeat the C-terminus, but it alternatively may be linked at the N-terminusas described above.

Suitably, the peptide is covalently linked to the therapeutic moleculethrough a linker if required. The linker may act as a spacer to separatethe peptide sequence from the therapeutic molecule.

The linker may be selected from any suitable sequence.

Suitably, the linker may be part of the peptide or the therapeuticmolecule.

Suitable linkers include, for example, a C-terminal cysteine residuethat permits formation of a disulphide, thioether or thiol-maleimidelinkage, a C-terminal aldehyde to form an oxime, a click reaction orformation of a morpholino linkage with a basic amino acid on the peptideor a carboxylic acid moiety on the peptide covalently conjugated to anamino group to form a carboxamide linkage.

Suitably, the linker is between 1-5 amino acids in length.

Suitably the linker is selected from any of the following sequences: BC,XC, C, GGC, BBC, BXC, XBC, X, XX, B, BB, BX and XB.

Any B or X may be replaced by another amino acid, such as Gly, Ala orPro or the side-chain of Glu or Asp, or any non-natural amino acidresidue, for example, 4-aminobutyryl (Aib) or isonicopecotinyl.

In one embodiment, the linker is β-alanine or glycine.

In one embodiment, the peptide is conjugated to the therapeutic moleculethrough a carboxamide linkage.

The linker of the conjugate may form part of the therapeutic molecule towhich the peptide is attached. Alternatively, the attachment of thetherapeutic molecule may be directly linked to the C-terminus of thepeptide. Suitably, in such embodiments, no linker is required.

Alternatively, the peptide may be chemically conjugated to thetherapeutic molecule. Chemical linkage may be via a disulphide, alkenyl,alkynyl, aryl, ether, thioether, triazole, amide, carboxamide, urea,thiourea, semicarbazide, carbazide, hydrazine, oxime, phosphate,phosphoramidate, thiophosphate, boranophosphate, iminophosphates, orthiol-maleimide linkage, for example.

Optionally, cysteine may be added at the N-terminus of a therapeuticmolecule to allow for disulphide bond formation to the peptide, or theN-terminus may undergo bromoacetylation for thioether conjugation to thepeptide.

Suitably the conjugate is capable of penetrating into cells and tissues,suitably into the nucleus of cells. Suitably into muscle tissues.

Suitably the conjugate is capable of penetrating into cells of thecentral nervous system.

Suitably the conjugate is capable of crossing the blood brain barrier,suitably into many compartments of the central nervous system.

Suitably the conjugate is capable of crossing the blood brain barrier,suitably into the cortex, brainstem, cerebellum, cervical, thoracic, andlumber compartments of the central nervous system.

Pharmaceutical Composition

The conjugate of the invention may formulated into a pharmaceuticalcomposition.

Suitably, the pharmaceutical composition may further comprise apharmaceutically acceptable diluent, adjuvant or carrier.

Suitable pharmaceutically acceptable diluents, adjuvants and carriersare well known in the art.

As used herein, the phrase “pharmaceutically acceptable” refers to thoseligands, materials, formulations, and/or dosage forms which are, withinthe scope of sound medical judgment, suitable for use in contact withthe tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier”, as used herein, refersto a pharmaceutically acceptable material, formulation or vehicle, suchas a liquid or solid filler, diluent, excipient, solvent orencapsulating material, involved in carrying or transporting theconjugate from one organ or portion of the body, to another organ orportion of the body. Each carrier must be “acceptable” in the sense ofbeing compatible with the other components of the composition e.g. thepeptide and therapeutic molecule, and not injurious to the individual.Lyophilized compositions, which may be reconstituted and administered,are also within the scope of the present composition.

Pharmaceutically acceptable carriers may be, for example, excipients,vehicles, diluents, and combinations thereof. For example, where thecompositions are to be administered orally, they may be formulated astablets, capsules, granules, powders, or syrups; or for parenteraladministration, they may be formulated as injections (intramuscular,subcutaneous, intramedullary, intrathecal, intraventricular,intravenous, intravitreal), drop infusion preparations, orsuppositories. These compositions can be prepared by conventional means,and, if desired, the active compound (i.e. conjugate) may be mixed withany conventional additive, such as an excipient, a binder, adisintegrating agent, a lubricant, a corrigent, a solubilizing agent, asuspension aid, an emulsifying agent, a coating agent, or combinationsthereof.

It should be understood that the pharmaceutical compositions of thepresent disclosure can further include additional known therapeuticagents, drugs, modifications of compounds into prodrugs, and the likefor alleviating, mediating, preventing, and treating the diseases,disorders, and conditions described herein under medical use.

Medical Use

The conjugate comprising the peptide of the invention may be used as amedicament for the treatment of a disease.

The medicament may be in the form of a pharmaceutical composition asdefined above.

A method of treatment of a patient or subject in need of treatment for adisease condition is also provided, the method comprising the step ofadministering a therapeutically effective amount of the conjugate to thepatient or subject.

Suitably, the medical treatment requires delivery of the therapeuticmolecule into a cell, suitably into the nucleus of the cell.

Diseases to be treated may include any disease where improvedpenetration of the cell and/or nuclear membrane by a therapeuticmolecule may lead to an improved therapeutic effect.

Suitably, the conjugate is for use in the treatment of diseases of thecentral nervous system.

Conjugates comprising peptides of the invention are suitable for thetreatment of genetic diseases of the central nervous system. In asuitable embodiment, there is provided a conjugate according to thesecond aspect for use in the treatment of genetic diseases of thecentral nervous system.

Suitably, the conjugate is for use in the treatment of diseases causedby splicing deficiencies. In such embodiments, the therapeutic moleculemay comprise an oligonucleotide capable of preventing or correcting thesplicing defect and/or increasing the production of correctly splicedmRNA molecules.

Suitably the conjugate is for use in the treatment of any of thefollowing diseases: Duchenne Muscular Dystrophy (DMD), Bucher MuscularDystrophy (BMD), Menkes disease, Beta-thalassemia, dementia, Parkinson'sDisease, Spinal Muscular Atrophy (SMA), myotonic dystrophy (DM),Huntington's Disease, Hutchinson-Gilford Progeria Syndrome,Ataxia-telangiectasia, or cancer.

In one embodiment, the conjugate is for use in the treatment of SMA.

In one embodiment, there is provided a conjugate according to the secondaspect for use in the treatment of SMA.

Suitably, in such an embodiment, the therapeutic molecule of theconjugate is operable to reduce transcription of the truncated form ofthe SMN2 gene and increase transcription of the full-length form of theSMN2 gene.

Suitably, the therapeutic molecule of the conjugate is operable to do soby increasing inclusion of exon 7 during SMN2 transcription.

Suitably, the therapeutic molecule of the conjugate is operable toincrease expression of the SMN2 protein. Suitably the therapeuticmolecule of the conjugate increases expression of the SMN2 protein by upto 3 times compared with untreated subjects.

Suitably, the patient or subject to be treated may be any animal orhuman. Suitably, the patient or subject may be a non-human mammal.Suitably the patient or subject may be male or female.

Suitably, the conjugate is for administration to a subject systemicallyfor example by enteral, parenteral, intravenous, intra-arterial,intramuscular, intratumoural, oral or nasal routes.

In one embodiment, the conjugate is for administration to a subjectintravenously.

Suitably, the conjugate is for administration to a subject in a“therapeutically effective amount”, by which it is meant that the amountis sufficient to show benefit to the individual. The actual amountadministered, and rate and time-course of administration, will depend onthe nature and severity of the disease being treated. Decisions ondosage etcetera, are within the responsibility of general practitionersand other medical doctors. Examples of the techniques and protocols canbe found in Remington's Pharmaceutical Sciences, 20th Edition, 2000,pub. Lippincott, Williams & Wilkins.

Suitably, the conjugate is for administration to a subject at a dose ofbetween 0.01 mg/kg and 20 mg/kg, 0.05 mg/kg and 19 mg/kg, 0.1 mg/kg and18 mg/kg, 0.5 mg/kg and 17 mg/kg, 1 mg/kg and 16 mg/kg, 2 mg/kg and 15mg/kg, 5 mg/kg and 10 mg/kg, 10 mg/kg and 20 mg/kg, 12 mg/kg and 18mg/kg, 13 mg/kg and 17 mg/kg, for example or any value therebetween.

Nucleic Acids and Hosts

Peptides of the invention may be produced by any standard proteinsynthesis method, for example chemical synthesis, semi-chemicalsynthesis or through the use of expression systems.

Accordingly, the present invention also relates to the nucleotidesequences comprising or consisting of the DNA coding for the peptides,expression systems e.g. vectors comprising said sequences accompanied bythe necessary sequences for expression and control of expression, andhost cells and host organisms transformed by said expression systems.

Accordingly, a nucleic acid encoding a peptide according to the presentinvention is also provided.

Suitably, the nucleic acids may be provided in isolated or purifiedform.

An expression vector comprising a nucleic acid encoding a peptideaccording to the present invention is also provided.

Suitably, the vector is a plasmid.

Suitably the vector comprises a regulatory sequence, e.g. promoter,operably linked to a nucleic acid encoding a peptide according to thepresent invention. Suitably, the expression vector is capable ofexpressing the peptide when transfected into a suitable cell, e.g.mammalian, bacterial or fungal cell.

A host cell comprising the expression vector of the invention is alsoprovided.

Expression vectors may be selected depending on the host cell into whichthe nucleic acids of the invention may be inserted. Such transformationof the host cell involves conventional techniques such as those taughtin Sambrook et al [Sambrook, J., Russell, D. (2001) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, NY, USA].Selection of suitable vectors is within the skills of the personknowledgeable in the field. Suitable vectors include plasmids,bacteriophages, cosmids, and viruses.

The peptides produced may be isolated and purified from the host cell byany suitable method e.g. precipitation or chromatographic separatione.g. affinity chromatography.

Suitable vectors, hosts and recombinant techniques are well known in theart.

In this specification the term “operably linked” may include thesituation where a selected nucleotide sequence and regulatory nucleotidesequence are covalently linked in such a way as to place the expressionof a nucleotide coding sequence under the control of the regulatorysequence, as such, the regulatory sequence is capable of effectingtranscription of a nucleotide coding sequence which forms part or all ofthe selected nucleotide sequence. Where appropriate, the resultingtranscript may then be translated into a desired peptide.

BRIEF DESCRIPTION OF THE DRAWINGS

Certain embodiments of the present invention will now be described withreference to the following figures and tables in which:

FIG. 1: Shows the structure of Beta D-glucosyl serine residue;

FIG. 2: Shows a series of graphs showing increase in full length SMN2transcript expression analysed via qPCR in three skeletal muscles(tibialis anterior (TA), quadriceps (Quad) and gastrocnemius (Gastro)),three brain compartments (cortex, brainstem, and cerebellum) and threespinal cord compartments (cervical, thoracic, and lumbar) followingintravenous delivery of peptide 17-PMO conjugate and peptide 14-PMOconjugate in SMA adult mice at a dose of 2×15 mg/kg given approximately48 hours apart. Tissues were harvested 7 days post final administration.Expression levels shown are normalised to saline treated controls,represented by dashed line). Data represented as mean±SD (*P 0.05; **P0.005; ***P 0.0005; ****P 0.00005 by Student's t test in comparison tosaline treated controls);

FIG. 3: Cell screening of glucosylated peptide-PMO conjugates for SMN2exon 7 inclusion in human SMA patient fibroblast cell culture forpeptides 1-16 by semi quantitative PCR at 50 nM concentration;

FIG. 4: Cell screening of glucosylated peptide-PMO conjugates for SMN2exon 7 inclusion in human SMA patient fibroblast cell culture forpeptides 3-54 by qPCR at different concentrations (667 nM and 2 μM);

FIG. 5: Cell screening of glucosylated peptide-PMO conjugates for SMN2exon 7 inclusion in human SMA patient fibroblast cell culture forpeptides 55-72 by qPCR at different concentrations (667 nM and 2 μM);

FIG. 6: Cell screening of glycosylated peptide-PMO conjugates for SMN2exon 7 inclusion in human SMA patient fibroblast cell culture forpeptides 73-80 at different concentrations (4 μM, 2 μM, 1 μM, 500 nM,250 nM);

FIG. 7: Shows a series of graphs showing increase in full length SMN2transcript expression analysed via qPCR in three skeletal muscles(tibialis anterior (TA), quadriceps (Quad) and gastrocnemius (Gastro)),three brain compartments (cortex, brainstem, and cerebellum) and threespinal cord compartments (cervical, thoracic, and lumbar) followingintravenous delivery of peptide 17-PMO conjugate and currently availablepeptide 6-PMO conjugate. Expression levels shown are normalised tosaline treated controls, represented by dashed line). Data representedas mean±SD (*P≤0.05; ** P≤0.005; *** P≤0.0005; **** P≤0.00005 byStudent's t test in comparison to saline treated controls);

FIG. 8: Shows urinary KIM-1 and Lipocalin-2 (NGAL) levels normalised tocreatinine, two and seven days post-administration of 25 mg/kg singledose of currently available peptide 6 conjugate (Pip8b4-PMO) or peptide17 conjugate (DPEP 5.17-PMO);

Table 1: Shows the sequences of the peptides tested in vivoincorporating SEQ ID NO.s 1-7 and 14 and 17 with additional N and Cterminal modifications, including peptide and SEQ ID NO.s 1-7 ofcurrently available peptides, and peptide and SEQ ID NO.s 14 and 17 ofthe invention;

Table 2: Shows the sequences of peptides screened in vitro in SMApatient fibroblasts incorporating SEQ ID NO.s 1-80 with additional N andC terminal modifications, including peptide and SEQ ID NO.s 1-7 ofcurrently available peptides, and peptide and SEQ ID NO.s 8-80 of theinvention;

Table 3: Shows quantitative PCR data for levels of full-length SMN2transcripts generated in adult SMA mice treated with doses of 2×15 mg/kgof PMO conjugates with the peptides listed given approximately 48 hoursapart. Tissues were harvested 7 days post final administration and RNAcollected for qPCR analysis. Data represented as mean expression level(*P 0.05; **P 0.005; ***P 0.0005; ****P 0.00005 by Student's t-test incomparison to saline treated controls). P values for liver samples allexceeded P 0.00005 (not represented in table).

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of them mean “including but notlimited to”, and they are not intended to (and do not) exclude othermoieties, additives, components, integers or steps. Throughout thedescription and claims of this specification, the singular encompassesthe plural unless the context otherwise requires. In particular, wherethe indefinite article is used, the specification is to be understood ascontemplating plurality as well as singularity, unless the contextrequires otherwise.

Features, integers, characteristics, compounds, chemical moieties orgroups described in conjunction with a particular aspect, embodiment orexample of the invention are to be understood to be applicable to anyother aspect, embodiment or example described herein unless incompatibletherewith. All of the features disclosed in this specification(including any accompanying claims, abstract and drawings), and/or allof the steps of any method or 27 process so disclosed, may be combinedin any combination, except combinations where at least some of suchfeatures and/or steps are mutually exclusive.

The invention is not restricted to the details of any foregoingembodiments. The invention extends to any novel one, or any novelcombination, of the features disclosed in this specification (includingany accompanying claims, abstract and drawings), or to any novel one, orany novel combination, of the steps of any method or process sodisclosed. The reader's attention is directed to all papers anddocuments which are filed concurrently with or previous to thisspecification in connection with this application and which are open topublic inspection with this specification, and the contents of all suchpapers and documents are incorporated herein by reference.

EXAMPLES

1. Peptides Comprising a Beta D-Glucosyl Serine Residue.

The present inventors set out to find short peptides that, whenconjugated to an oligonucleotide therapeutic, might lead to significantand effective cell penetration and activity in all or most spinal cordand brain compartments.

Surprisingly, the present inventors discovered that inclusion of adirectly glycosylated amino acid (for example: S*, FIG. 1) into shortarginine rich peptide carriers, and in addition systemic injection intoadult SMA mice results in exon inclusion in 6 brain and spinal cordcompartments as well in skeletal muscles. This is demonstrated in thepresent examples with the synthesis of a series of arginine richpeptides having one or more β-D-glucosyl serine residues, and in somecases additional hydrophobic domains.

The present inventors synthesised a series of candidate peptides namedthe D-PEP5′ peptides. The first of which, peptide 14 (Table 1 and 2),comprises 10-Arg flanking arm domain sequences and an S* residueadjacent to a shortened 2-amino acid hydrophobic core of FL, and thesecond of which, peptide 17, comprises flanking 8-Arg arm domainsequences but has an S* residue adjacent to a single amino acidhydrophobic core of F (Table 1 and 2). A variety of currently availablepeptides were also synthesised for comparison with the inventive D-PEP5peptides. These are numbered as peptides 1-7 in table 1 and incorporatethe following peptide sequences:

(SEQ ID NO. 1) RXRRBRRXRYQFLIRXRBRXR (SEQ ID NO. 2) RXRRBRRXRQFLRXRBRXR(SEQ ID NO. 3) RXRRBRRFQILYRBRXR (SEQ ID NO. 4) RXRRBRRYQFLIRBRXR(SEQ ID NO. 5) RXRRBRRQFLRBRXR (SEQ ID NO. 6) RXRRBRRFLRBRXR(SEQ ID NO. 7) RXRRBRFQILYRBRXR

2. Increase in FLSMN2 Expression in Skeletal Muscles, Brain Compartmentsand Spinal Cord Compartments by Intravenous Delivery of Peptide 17-PMOor Peptide 14-PMO in SMO Mice.

The inventors then tested the in vivo administration of peptides 17 and14 and the currently available peptides 1-7 described above byconjugating the peptides to an antisense oligonucleotide therapeuticspecifically a PMO. The antisense oligonucleotide was specificallydirected at treating SMA by increasing full length SMN2 transcriptproduction.

Smn1tm1Hung/wt; SMN2tg/tg and Smn1wt/wt; SMN2tg/tg mice were treated at7-8 weeks of age with two administrations given two days (approximately48 hours) apart of 15 mg/kg peptide 17-PMO, peptide 14-PMO conjugates orsaline only (control). Tissues were harvested one week postadministration. Quantitative PCR was performed on extracted RNA toanalyse the amount of full-length SMN2 transcripts (in relation to totalSMN2 transcripts), see FIG. 2. Each treatment groups was normalised totheir saline controls, represented by the dashed line. Statisticalsignificance determined by Student's t-test *p 0.05, **p 0.005, ***p0.0005, ****p 0.00005.

Quantitative PCR analysis of brain, spinal cord and skeletal muscletissues showed a significant increase in the amount of full-length SMN2(FLSMN2) transcripts in several areas of the brain and spinal cord (FIG.2). Treated skeletal muscles gave around a 3-fold increase infull-length SMN2 expression.

Quantitative data for levels of cell penetration of the peptides invivo, measured by an increase in full-length SMN2 transcripts, are shownin Table 3 for the peptide 14 conjugate and the peptide 17 conjugatewhen compared with currently available peptide conjugates to the sameantisense oligonucleotide. Data could not be obtained for TA skeletalmuscle after treatment with peptide 6 conjugate in this experiment.However, the data demonstrate that cell penetration is increased inseveral compartments of the central nervous system and the skeletalmuscle for peptide 14 and peptide 17 of the invention when compared withcurrently available peptides 1-7.

Specifically, peptide 6 can be compared with peptide 17 as both share avery similar sequence with the exception that peptide 17 of theinvention has a glycosylated serine residue in place of a hydrophobicresidue. Peptide 2 can be compared directly with peptide 14 as bothshare the same sequence with the exception that peptide 14 of theinvention has a glycosylated serine residue in place of a glutamineresidue and contains an additional linker beta-alanine. Peptide 17 showsincreased cell penetration in the cortex, cerebellum, cervical, andthoracic compartments of the CNS, and increases in skeletal musclepenetration compared with peptide 6 of up to 60.2%. Peptide 14 showsincreased cell penetration in the cortex, brainstem, cerebellum,thoracic and lumbar compartments of the CNS when compared with peptide 2of up to 59.7%.

Further data was obtained to show a direct comparison of peptide 6 whichis a currently available carrier peptide (designated Pip8b4) withpeptide 17; a carrier peptide of the invention. Two systemic intravenousdoses of 15 mg/kg of peptide-PMO conjugates were administered 48 hoursapart in adult intermediate SMA mice (non-symptomatic SMN2 transgenicmice) and critical central and peripheral tissues were harvested 7 dayspost-final administration similar to the method described above. Resultsare shown in FIG. 7. Levels of exon 7 included transcripts (FLSMN) wereassayed via qPCR as explained above. The levels of full lengthtranscript were increased by treatment with peptide 17 for each tissue,exceeding the 1 fold increase threshold of FLSMN in all tissues (centralspinal cord and peripheral skeletal muscle and liver). Moreover, peptide17 of the invention showed equivalent or improved activity in thecritical central brain tissues of the cerebellum and cortex whencompared with the closest currently available carrier peptide; peptide6. This indicates that the peptides of the invention are more effectivethan those that are currently available. This further indicates that theinventive peptides are effective as a therapy.

3. Toxicology Profile of Glucosylated Peptide-PMO Conjugates

The inventors then further tested the toxicology of peptide 17 of theinvention (designated DPEP5.17) in comparison with the currentlyavailable peptide; peptide 6 (designated Pip8b4).

Urinary and serum markers of kidney and liver toxicity were measuredfollowing a single dose administration of saline or the relevantpeptide-PMO conjugate to 8 week old adult C57/BL10 female mice (N=6 pergroup). A bolus IV (tail vein) injection was administered and urinecollected Day 2 and Day 7 after administration. Animals were thenscarified prior to necropsy in which kidney, liver, diaphragm, heart,TA, gastric and serum were collected. Urine clinical indicators: KIM-1,NGAL were measured by ELISA (R&D cat # MKM100) with samples diluted tofit within standard curve. Values were normalised to urinary creatininelevels (Harwell) to account for urine protein concentration. Results areshown in FIG. 8.

Group 1—0.9% Saline

Group 2—25 mg/kg Pip8b4-PMO SMN

Group 3—25 mg/kg DPEP5.17-PMO SMN

By 7 days post administration, the levels of both KIM-1 and NGAL werelower in mice which had received the inventive peptide 17 than thosewhich had received peptide 6. Furthermore, 2 days after administration,the levels of the marker KIM-1 were far lower in mice which had receivedthe inventive peptide 17 than those which had received peptide 6. Thisindicates that the peptides of the invention have a better toxicologyprofile than those that are currently available, and therefore a lowertoxicity than the currently available peptides. This indicates that theinventive peptides are suitable for use as a therapy.

4. Cell Screening of Glucosylated Peptide-PMO Conjugates for ExonInclusion in Human SMA Patient Fibroblast Cell Culture of Peptides 8-54.

In order to generate further peptide candidates containing aglycosylated Serine residue for in vivo evaluation, the inventorssynthesized a range of similarly glycosylated peptides, conjugated themto the same PMO therapeutic and screened the resultant P-PMO conjugatesin human SMA patient fibroblast cell culture, which assesses theirability to enter cell nuclei to give exon inclusion.

These further peptides are shown in Table 2, as peptides 8-80. Such acellular screen provides candidates that are competent for enteringcells and effecting SMN2 exon inclusion of an attached PMO. Someimportant conclusions could be reached as to what changes affected exoninclusion activity in cells. In addition, P-PMO conjugates were checkedby MALDI-TOF spectrometry for their serum stability in mouse serum.

In the first screen (FIG. 3, D-PEP5 peptides 8-16 and FIG. 4, D-PEP5peptides 14, and 17-54), a study of 10-Arginine peptides related to thefirst DPEP5 peptide number 14 was undertaken. It was found thatreplacement of all X residues by B (peptide 18) had no significanteffect on cell activity. Replacement of S*FL by S in the core region(peptide 44) had no significant effect on cell activity. Replacement byS*F resulted in only a slightly reduced activity (peptide 43). The keyresult is that a glycosylated residue and core domain sequence such asS*FL could be placed in a variety of positions and contexts in thesequence without a large loss in cell activity (peptides 33, 34, 36, and37). S*FL placed close to the C-terminus (peptide 48) was almost asactive as the first peptide, peptide 14.

It was found that exon inclusion activity is generally reduced as thenumber of Arginine residues is reduced in the peptide. For example, the9-Arginine sequences peptide 24 and peptide 29 were less active than thefirst peptide 14, 8-Arginine sequences less active than these and the7-Arginine peptide 26 slightly less active only.

5. Cell Screening of Glucosylated Peptide-PMO Conjugates for ExonInclusion in Human SMA Patient Fibroblast Cell Culture of D-PEP5Peptides 55-72

Following the screen of D-PEP5 peptides 8-54, a second screen in cellswas carried out (FIG. 5) following synthesis of still furtherglucosylated D-PEP5 peptides 55-72 as shown in (Table 2). For the8-Arginine peptides, there was no significant alteration in activitylevels when FS* (peptides 56 and 57) was replaced by S* (peptides 58 and59). Double S* (peptide 67) and Triple S* (peptide 68) peptides retainedgood activity in cells. Amongst the 6-Arginine peptides, by and largethese had acceptable activity as PMO conjugates. However, P-PMOconjugates with peptide 70 and peptide 71 showed remarkably goodactivity.

6. Cell Screening of Glycosylated Peptide-PMO Conjugates for ExonInclusion in Human SMA Patient Fibroblast Cell Culture of D-Pep5Peptides 73-80.

Following the screen of the D-PEP5 peptides 8-72 a third screen in cellswas carried out (FIG. 6) following synthesis of still furtherglycosylated peptides 73-80 as shown in (Table 2). For the 8-Argininepeptides, there was no significant alteration in activity levels when S*was replaced by different glycosylated serine and asparagine moietiesN*, and S²*-S⁶* (peptide 75 to peptide 79). Improved activity was seenfor peptide 74 carrying a glycosylated unnatural D-serine residue andthereby preventing protease induced cleavage of the peptide. Furtherstabilisation of the peptide by introducing a unnatural amino acid(peptide 73, Z=Tic=1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid)within the hydrophobic core-region greatly improved alteration inactivity levels and even showed good activity at low doses.

The three in vitro screens performed evaluate the ability of theinventive peptides to enter cells and effect SMN2 exon 7 inclusion inhuman SMA patient fibroblast cell culture and is a pre-requisite beforein vivo evaluation.

In summary, the present inventors have synthesised and demonstrated theimproved effectiveness of a series of peptides having one or moredirectly glucosylated amino acid residues present within an argininerich structure in penetrating into compartments of the CNS and muscularsystem for use as carriers for therapeutic molecules with a lowertoxicity.

Materials and Methods

Reagents and General Methods

9-Fluorenylmethoxycarbonyl (Fmoc) protected L-amino acids, and theFmoc-β-Ala-OH preloaded Wang resin (0.19 mmol g⁻¹) were obtained fromMerck (Hohenbrunn, Germany). HPLC grade acetonitrile, methanol andsynthesis grade N-methyl-2-pyrrolidone (NMP) were from Fisher Scientific(Loughborough, UK). Peptide synthesis grade N,N-dimethylformamide (DMF),benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium (PyBOP) and diethylether were obtained AGTC Bioproducts (Yorkshire, UK). Piperidine andtrifluoroacetic acid (TFA) were obtained from Alfa Aesar (Heysham,England). PMO was purchased from Gene Tools Inc. (Philomath, USA). Fetalbovine serum, mouse serum and Superscript III Platinium One-Step qRT-PCRKit and Platinum PCR SuperMix High Fidelity were obtained fromThermofisher Scientific (Waltham, US). iScript cDNA Synthesis Kit wasobtained from Biorad (Hercules US). All other reagents were obtainedfrom Sigma-Aldrich (St. Louis, Mo., USA) unless otherwise stated.MALDI-TOF mass spectrometry was carried out using a Voyager DE ProBioSpectrometry workstation. A stock solution of 10 mg mL⁻¹ ofα-cyano-4-hydroxycinnamic acid or sinapinic acid in 60% acetonitrile inwater containing 0.1% TFA was used as matrix.Fmoc-L-Ser(Ac₄-β-D-Glc)—OH═S*, Fmoc-D-Ser(Ac₄-β-D-Glc)—OH═S¹*,Fmoc-L-Ser(Ac₄-β-L-Glc)—OH═S²*, Fmoc-L-Ser(Ac₄-β-D-Gal)—OH═S⁶*,Fmoc-L-Ser(Ac₄-α-D-Man)—OH═S³*, 2—N-Fmoc-4—N-[Ac₄-β-D-Glc)-L-Asn-OH═N*,Fmoc-L-Ser(β-D-Lac(Ac)₇)—OH═S⁴*, were synthesized as previouslydescribed (15-18).

Synthesis of Peptides on 100 μMol Scale

Peptides were synthesized on a 100 μmol scale using a CEM Liberty™microwave Peptide Synthesizer (Buckingham, UK) and Fmoc chemistryfollowing manufacturer's recommendations. The side chain protectinggroups used were labile to trifluoroacetic acid treatment and thepeptide was synthesized using a 5-fold excess of Fmoc-protected aminoacids (0.25 mmol) that were activated using PyBOP (5-fold excess) in thepresence of DIPEA or with DIC|Oxyma. Piperidine (20% v/v in DMF) wasused to remove N-Fmoc protecting groups. The coupling was carried outonce at 75° C. for 5 min at 60-watt microwave power except for arginineand the glycosylated amino acid residues, which were coupled twice each.

Histidine and cysteine residues were coupled once at 50° C. for 5 min at60-watt microwave power. Each deprotection reaction was carried out at75° C. twice, once for 30 sec and then for 3 min at 35-watt microwavepower. Once synthesis was complete, the resin was washed with DMF (3×50mL) and the N-terminus of the solid phase bound peptide was acetylatedwith acetic anhydride in the presence of DIPEA. The peptide was cleavedfrom the solid support by treatment with a cleavage cocktail consistingof trifluoroacetic acid (TFA): 3,6-dioxa-1,8-octanedithiol (DODT): H₂O:triisopropylsilane (TIPS) (94%: 2.5%: 2.5%: 1%, 10 mL) ortrifluoroacetic acid (TFA): H₂O: m-cresol: triisopropylsilane (TIPS)(94%: 2.5%: 2.5%: 1%, 1 mL) or trifluoroacetic acid (TFA): H₂O:triisopropylsilane (TIPS) (96.5%: 2.5%: 1%, 1 mL) for 2 h at roomtemperature for 2-3 h at room temperature. Excess TFA was removed byblowing N2 through the peptide solution. The cleaved peptide wasprecipitated via the addition of ice-cold diethyl ether and centrifugedat 3000 rpm for 5 min. The peptide pellet was washed in ice-cold diethylether thrice. The crude peptide was dissolved in water, analyzed andpurified by RP-HPLC on Phenomenex Jupiter column (21.2×250 mm, C18, 10μm) at a flow rate of 20 mL/min with the following gradient (A: 0.1%TFA, B: 90% CH₃CN, 0.1% TFA) 0-2 min 5% B 2-35 min 5%-60% B 35-40 min60%-90% B used. The fractions containing the desired peptide werecombined and lyophilized to give the product as a white solid.

Synthesis of a Library of Peptide Variants on 5 μMol Scale

Each peptide was prepared on a 5 μmol scale using an Intavis ParallelPeptide Synthesizer using Fmoc-Gly-HMP-Tentagel resin (0.2 mmol g⁻¹) orFmoc-β-Ala-Wang Chemmatrix resin (0.3 mmol g⁻¹) by applying standardFmoc chemistry and following manufacturer's recommendations. Doublecoupling steps were used with a PyBOP/NMM coupling mixture followed byacetic anhydride capping after each step. The peptides were cleaved fromthe solid support by treatment with a cleavage cocktail consisting oftrifluoroacetic acid (TFA): 3,6-dioxa-1,8-octanedithiol (DODT): H₂O:triisopropylsilane (TIPS) (94%: 2.5%: 2.5%: 1%, 1 mL) or trifluoroaceticacid (TFA): H₂O: m-cresol: triisopropylsilane (TIPS) (94%: 2.5%: 2.5%:1%, 1 mL) or trifluoroacetic acid (TFA): H₂O: triisopropylsilane (TIPS)(96.5%: 2.5%: 1%, 1 mL) for 2 h at room temperature. After peptiderelease, excess TFA was removed by blowing N2 gas into the TFA solution.The crude peptide was precipitated by the addition of cold diethyl ether(12 mL) and centrifuged at 2500 rpm for 5 min. The crude peptide pelletwas washed thrice by cold diethyl ether (3×12 mL). The crude peptide wasdissolved in 1500 μL H₂O: CH₃CN mixture and purified by RP-HPLC using aPhenomenex Jupiter column (10×250 mm, C18, 10 mm) at a flow rate 5mL/min with the following gradient (A: 0.1% TFA, B: 90% CH₃CN, 0.1% TFA)0-2 min 5% B 2-35 min 5%-60% B 35-40 min 60%-90% B. The fractionscontaining the desired peptide were combined and lyophilized to yieldthe peptide as a white solid (see Table 1 for yields).

Synthesis of PMO-Peptide Conjugates

The following PMO antisense sequences targeting the human SMN2-gene wereused.

ISS-N1-20 mer: (SEQ ID NO. 147) ATT CAC TTT CAT AAT GCT GGISS-N1-25 mer: (SEQ ID NO. 148) GTA AGA TTC ACT TTC ATA ATG CTG G

The peptide was conjugated to the 3′-end of the PMO through itsC-terminal carboxyl group. This was achieved using 2.5 and 2 equivalentsof HBTU and HOAt in NMP respectively in the presence of 2.5 equivalentsof DIPEA and 2.5 fold excess of peptide over PMO dissolved in DMSO wasused.

PMO Peptide-COOH HBTU HOAt DIPEA 10 mM 100 mM 300 mM 300 mM 1 eq. 2.5eq. 5.75 eq. 5.75 eq. 5.75 eq. 100 nmol 250 nmol 575 nmol 575 nmol 575nmol 10 μl 2.5 μl 1.92 μl 1.92 μl 0.11 μl

To a solution of peptide (250 nmol) in N-methylpyrrolidone (NMP, 2.5 μL)were added HBTU (1.92 μL of 0.3 M in NMP), HOAt in (1.92 μL of 0.3 MNMP), DIPEA (0.1 μL) and PMO (10 μL of 10 mM in DMSO). The mixture wasleft for 2-3 h at 40° C. and the sugar protecting groups were globallydeprotected by the addition of 10 μl hydrazine hydrate. After 10 min thedeprotection reaction was quenched by the addition of ice cold 5% AcOH(1000 μL). This solution was then purified by Ion exchangechromatography using a Resource S HR-161100 column at a flow rate 6mL/min with the following gradient (A: 25 mM phosphate buffer pH 7 with25% ACN, B: 25 mM phosphate buffer pH 7 with 25% CH₃CN and 1M NaCl) 0-2min 0% B 2-20 min 0%-75% B 20-23 min 100% B, 23-28 100% A. The fractionscontaining the desired compound were desalted (Amicon 15 Ultracel, MWCO3 kDa, EMD Millipore) and lyophilized.

Mouse Serum Stability Experiments

10 nmol of lyophilized P-PMO was dissolved in 100 μl mouse serum andincubated at 37° C. for different time periods. Samples were dilutedwith 300 μl guanidinium-HCl solution (10 ml of 1M guanidinium-HClcontaining 1 tablet complete mini protease inhibitor cocktail (Roche,Basel, Switzerland) and with 600 μl ice cold acetonitrile andcentrifuged at 14.000 rpm for 3 min. The supernatant was collected andanalysed by MALDI TOF MS and ion exchange chromatography.

Cell Culture

GM03813 patient fibroblast cells were cultured in T75 flasks at 37° C.,under 5% CO2 in Dulbecco's modified Eagle's medium (DMEM with Glutamax,Thermofisher) supplemented with 10% heat-inactivated fetal bovine serum(FBS Gold, PAA laboratories), 1% penicillin-streptomycin-neomycinantibiotic mixture (PSN, Gibco).

Cytotoxicity

GM03813 patient fibroblasts were seeded out at 1250 cells|well in 100 μlDulbecco's modified Eagle's medium (DMEM) with GlutaMAX and 10% fetalbovine serum (FBS) (Life Technologies, Inc.) in 96 well plates, andincubated for 16 hours in a cell culture incubator (37° C., 5% CO2, 100%rel. humidity). Afterwards the media was removed and cells were washedonce with Opti-Mem and treated with different concentrations of PPMO inOpti-Mem in duplicate for 4 hours at 37° C. Subsequently thetransfection mixture was replaced by normal culture media and cells wereallowed to grow overnight. On the next day 20 μl of MTS Cell Viabilityassay (Promega) was added to the wells and incubated for 3 hours beforemeasurement at 490 nm were taken. The cell viability percentage wasdetermined by normalizing the average absorbance of triplicate samplesto the mean of untreated samples.

qPCR Analysis of SMN2 Full Length and A7 mRNA in Cultured Cells

GM03813 (Coriell Institute) derived from SMA type I patient fibroblastwere seeded out at 2500 cells/well in 100 μl Dulbecco's modified Eagle'smedium (DMEM) with GlutaMAX and 5% fetal bovine serum (FBS) (LifeTechnologies, Inc.) in 96 well plates, and incubated for 16 h in a cellculture incubator (37° C., 5% CO₂, 100% rel. humidity). On the next daycells were then treated with 10 μl P-PMO at different concentrations (inwater) in duplicate for 24 hours. After removal of the supernatant,cells were washed once with PBS-buffer and were lysed in lysis buffer(10 mM Tris, 3 mM MgCl₂, 1 mM CaCl₂), 1% Triton X-100, 200 u/ml DNase Iand 200 u/ml Proteinase K) for 10 min. Afterwards the lysate weretransferred into a 96-well plate (Eppendorf twintec) and incubated at75° C. for 15 min and subsequently cooled to 4° C. and used immediately.The mRNA levels of SMN2 FL, SMN2 Δ7 and GAPDH were quantified usingTaqman-based qRT-PCR (Superscript® μl Platinium® One Step qRT-PCR,Thermo Fisher) and SMN2 specific Primers and probes (purchased from IDTIntegrated DNA Technologies). (19) SMN2 FL and 47 mRNAs were normalizedto GAPDH.

Endpoint qPCR Analysis of SMN2 Full Length and A7 mRNA in Cultured Cells

GM03813 (Coriell Institute) derived from SMA type I patient fibroblastswere seeded out at 1×10⁵ cells/well in 2000 μl Dulbecco's modifiedEagle's medium (DMEM) with GlutaMAX and 10% fetal bovine serum (FBS)(Life Technologies, Inc.) in 6 well plates, and incubated for 2 days(until cells reach a confluency greater than 90%) in a cell cultureincubator (37° C., 5% CO2, 100% rel. humidity). Afterwards cells werewashed once with PBS and Opti-Mem and cells were then treated with 1000μl of PPMO in Opti-Mem in duplicates for 4 h. The transfection mediumwas then replaced with DMEM supplemented with 10% fetal bovine serum and1% PSN and the cells incubated for a further 20 hr at 37° C. Cells werewashed with PBS and 0.5 mL of TRI RNA (Sigma) isolation reagent wasadded to each well. Cells were frozen at −80° C. for 1 h.

RNA Extraction and Nested RT-PCR Analysis

Total cellular RNA was extracted using TRI reagent with an extra furtherprecipitation with ethanol. The purified RNA was quantified using aNanodrop® ND-1000 (Thermo Scientific). The RNA (500 ng) was used as atemplate for 2 step RT-PCR using iScript cDNA Synthesis Kit (Biorad,Hercules US) and Platinum PCR SuperMix High Fidelity (ThermofisherScientific (Waltham, US). Primers (Forward: 5′-CTC CCA TAT GTC CAG ATTCTC TT-3′ (SEQ ID NO.149) and Reverse: 5′-CTA CAA CAC CCT TCT CAC AG-3′(SEQ ID NO.150) were used to amplify full-length (505 bp) and Δ7 SMN2(451 bp) from cDNA. The products were amplified semi-quantitativelyusing 30 PCR cycles (94° C. for 30 s, 55° C. for 30 s and 72° C. for 30s). All PCR products were checked by electrophoresis on 2% agarose gels.

Data Analysis

The images of agarose gels were taken on a Molecular Imager ChemiDoc™XRS⁺ imaging system (BioRad, UK) and the images were analysed usingImage Lab (V4.1). Microsoft Origin was used to analyse and plot theexon-inclusion assay data, which were expressed as the percentage of 47SMN2 transcript from at least three independent experiments.

Animal Models

Experiments were carried out in the Biomedical Sciences Unit, Universityof Oxford according to procedures authorized by the UK Home Office.Experiments were performed in SMA like mouse strainFVB.Cg-Smn1^(tm1Hung)Tg(SMN2)2Hung/J, Jackson Laboratory stock number5058 (28). The line was maintained and heterozygous mice(Smn1^(tm1Hung/wt);SMN2^(tg/tg)) were generated as previously described(21). Two doses of 15 mg/kg (given 2 days, about 48 hours apart) werediluted in 0.9% saline (Sigma) and administered via intravenous tailvein at a volume of 5 μl per gram body weight. Tail vein administrationswere performed after warming mice at 32° C. Mice were then restrained inapproved apparatus and peptide-PMO conjugates IV administered withoutanaesthetics. Administered mice were allowed to recover in heat box.Saline treated control animals were selected from littermates andhandled in the same manner as the treated animals to control forpotential changes in SMN expression due to stress. Tissues wereharvested 7 days post final-administration. The tissues harvestedincluded: liver, quadriceps, gastric, TA, brain, brainstem, cerebellum,and spinal cord. Spinal cord was divided into cervical, thoracic andlumbar regions. Tissues were snap frozen in liquid nitrogen and storedat −80° C.

RNA Extraction and cDNA Synthesis

RNA extraction from tissues was carried out using TRIZOL extraction,however another suitable product is Qiagen RNeasy® Mini Kit (Qiagen#74104), following manufacturer's instructions. One microgram of RNAtemplate was used in a 20 μl reverse transcription reaction using ABIHigh Capacity cDNA Reverse Transcription Kits (Invitrogen, Carlsbad,Calif.).

SMN mRNA QPCR

RNA extraction from harvested tissues was carried out using TRIZOLextraction, however another suitable product is Qiagen RNeasy® Mini Kit.One microgram of RNA template was used in a 20 μl reverse transcriptionreaction using ABI High Capacity cDNA Reverse Transcription Kits(Invitrogen, Carlsbad, Calif.). Synthesized cDNA was diluted 1:5 withddH2O and used at 20 ng per 20 μl QPCR reaction using Power SYBR® GreenMaster Mix (Life Technologies). Real time QPCR is performed and analysedon Applied Biosystems® StepOnePlus™ real-time PCR system (LifeTechnologies). Full length SMN2 transcript (FLSMN2) was amplified usinggene-specific primers Exon 6 Fwd: 5′-GCT TTG GGA AGT ATG TTA ATT TCA-3′(SEQ ID NO.151), Exons 7-8 Rev: 5′-CTA TGC CAG CAT TTC TCC TTA ATT-3′(SEQ ID NO.152). SMN2 transcripts representing both FL and Δ7 mRNA wereamplified using gene specific primers (Exon 2a Fwd: 5′-GCG ATG ATT CTGACA TTT GG-3′ (SEQ ID NO.153), Exon 2b Rev: 5′-GGA AGC TGC AGT ATT CTTCT-3′ (SEQ ID NO.154). Cycle conditions: 95° C. for 10 minutes holdingstage, followed by 40 cycles of 15 seconds at 95° C. and 1 minute at 60°C. The melt curve was determined from 60° C. to 95° C. in 0.6° C. steps.Transcripts were normalized to Polymerase (RNA) II polypeptide J (PolJ)levels. PolJ Forward: 5′-ACCACACTCTGGGGAACATC-3′ (SEQ ID NO.155); PolJReverse: 5′-CTCGCTGATGAGGTCTGTGA-3′ (SEQ ID NO.156). ΔΔCt was calculatedas the difference between the ΔCt values, determined with the equation(PCR efficiency)^(−Ct). The PCR efficiency was determined by LinRegPCRsoftware (22,23). One-way ANOVA followed by Tukey's multiple comparisonstest was performed using GraphPad Prism version 6.05 for Windows(GraphPad Software, La Jolla Calif. USA, www.graphpad.com).

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TABLE 1 Peptide number incorporating corresponding SEQ ID. NO. Sequence 1 Ac-RXRRBRRXRYQFLIRXRBRXR-B  2 Ac-RXRRBRRXRQFLRXRBRXR-B  3Ac-RXRRBRRFQILYRBRXR-B  4 Ac-RXRRBRRYQFLIRBRXR-B  5 Ac-RXRRBRRQFLRBRXR-B 6 Ac-RXRRBRRFLRBRXR-B  7 Ac-RXRRBRFQILYRBRXR-B 14Ac-RXRRBRRXRS*FLRXRBRXR-BB 17 Ac-RXRRBRR FS*RBRXR-B

TABLE 2 Peptide number incorporating corresponding SEQ ID. NO. Sequence 1 Ac-RXRRBRRXRYQFLIRXRBRXR-B  2 Ac-RXRRBRRXRQFLRXRBRXR-B  3Ac-RXRRBRRFQILYRBRXR-B  4 Ac-RXRRBRRYQFLIRBRXR-B  5 Ac-RXRRBRRQFLRBRXR-B 6 Ac-RXRRBRRFLRBRXR-B  7 Ac-RXRRBRFQILYRBRXR-B  8Ac-RXRRBRRXRQFLRXRBRXRS*-B  9 Ac-RXRRBRRXRQFLRXRS*RXR-B 10Ac-RXRRS*RRXRQFLRXRBRXR-B 11 Ac-S*RXRRBRRXR QFL RXRBRXR-B 12Ac-RXRRBRRXR S*QFLS*RXRBRXR-B 13 Ac-RXRRBRRXR S*QFLRXRBRXR-B 14Ac-RXRRBRRXR S*FLRXRBRXR-BB 15 (Ac-S*BRKBRKRBBR)₂K-B 16Ac-GFTGPLS*BRKBRKRBBR)₂K-B 17 Ac-RXRRBRR FS*RBRXR-B 18Ac-RBRRBRRBR S*FL RBRBRBR-G 19 Ac-RGRRGRRGR S*FL RGRGRGR-G 20Ac-RPRRPRRPR S*FL RPRPRPR-G 21 Ac-RHypRRHypRRHypRS* FLRHypRHypRHypR-GHyp = hydroxy-proline 22 Ac-RARRARRAR S*FL RARARAR-G 23Ac-RCyRRCyRRCyRS*FLR CyR CyR CyR-G Cy = 1-(amino)cyclohexanecarboxylicacid 24 Ac-RRBRRBRS*FLRBRBRBR-G 25 Ac-RBRRBRS*FLRBRBRBR-G 26Ac-RRBRS*FLRBRBRBR-G 27 Ac-RBRS*FLRBRBRBR-G 28 Ac-RS*FLRBRBRBR-G 29Ac-RBRRBRRBRS*FLRBRBR-G 30 Ac-RBRRBRRBRS*FLRBR-G 31 Ac-RBRRBRRBRS*FLR-G32 Ac-RBRRBRRBRS*FL-G 33 Ac-RBRRBRRBRS*FLRBRBRR-G 34Ac-RBRRBRRBRS*FLRBRRR-G 35 Ac-RBRRBRRBRS*FLRRRR-G 36Ac-RBRRBRRRS*FLRBRBRBR-G 37 Ac-RBRRRRRS*FLRBRBRBR-G 38Ac-RRRRRRS*FLRBRBRBR-G 39 Ac-RBRRBRRRS*FLRRBRR-G 40 Ac-RBRRRRRS*FLRRRR-G41 Ac-RRRRRRS*FLRRRR-G 42 Ac-RGRRS*GRRGRS*FLRGGRBRGGR-G 43Ac-RXRRBRRXRS*FRXRBRXR-G 44 Ac-RXRRBRRXRS*RXRBRXR-G 45Ac-RXRRBRRS*FQILYRBRXR-G 46 Ac-RXRRBRRS*FLRBRXR-G 47Ac-RXRRBRRXRS*FLRXRBRXRS*FL-G 48 Ac-RXRRBRRXRRXRBRXRS*FL-G 49Ac-RXRRS*RRXRS*FLRXRS*RXR-G 50 Ac-RXRRBRRXRS*FQRXRBRXR-G 52Ac-RXRRBRRXRS*WFRXRBRXR-G 53 Ac-RXRRBRRXRS*QFRXRBRXR-G 54Ac-RXRRBRRXRS*FQRXRBS*YQFLIRXR-G 55 Ac-RXRRBRRS*RBRXR-G 56Ac-RXRRFS*RRBRBRXR-G 57 Ac-R FS*RRBRRBRBRXR-G 58 Ac-RXRRS*RRBRBRXR-G 59Ac-RS*RRBRRBRBRXR-G 60 Ac-RXRRBRRBRS*RXR-G 61 Ac-RXRRBRRBRBRS*R-G 62Ac-RXRRBRFS*RBR-G 63 Ac-RXRRBRS*RBR-G 64 Ac-RXRBRRS*RBR-G 65Ac-RRBRRS*RBR-G 66 Ac-HXHRBRRXRS*RXHBHXR-G 67 Ac-RXRRBRRS*S*RBRXR-G 68Ac-RXRRBRRS*S*S* RBRXR-G 69 Ac-RXRRS*RRS*RS*RXR-G 70 Ac-RBRBRS*RBRBR-G71 Ac-RXRXRS*RXRXR-G 72 Ac-RXRRBS*BRBRBR-G 73 Ac-RXRRBRRZS*RBRXR-B(Z = Tic = 1,2,3,4- tetrahydroisoquinoline-3- carboxylic acid) 74Ac-RXRRBRRFS ¹ *RBRXR-B, S ¹ * = D-Ser[D-Glc] 75 Ac-RXRRBRRFS ²*RBRXR-B, S ² * = L-Ser[L-Glc] 76 Ac-RXRRBRRFS ³ *RBRXR-B, S ³* = (L-Ser[D-Man] 77 Ac-RXRRBRRFS ⁴ *RBRXR-B, S ⁴ * = L-Ser[D-Lac] 78Ac-RXRRBRRFN*RBRXR-B, N* = L-Asn[D-GlcNac] 79 Ac-RXRRBRRFS ⁶ *RBRXR-B, S⁶ * = L-Ser[Gal]* 80 Ac-RAzRRAzRRZS*RAzRAzR-B (Z = Tic = 1,2,3,4-tetrahydroisoquinoline- 3-carboxylic acid,Az = 3-azetidine-carboxylic acid)

TABLE 3 Peptide No. 1 2 3 4 5 6 7 17 14 Central Nervous System Cortex1.29** 1.16* 0.93 1.1 1.11 1.13 1.01 1.27* 1.50* Brainstem 1.48***1.36*** 0.86 0.98 1.22 1.38** 1.11 1.2 1.97*** Cerebellum 1.25* 1.15*1.025 0.96 1 1.05 0.97 1.19* 1.26 Cervical 1.50*** 1.78** 0.92 1.14*1.24* 0.88** 1.26 1.41* 1.23** Thoracic 1.45*** 1.49* 1 1.12 1.23 1.37*1.33 1.39** 2.38** Lumbar 1.28*** 1.48** 1.1 1 1.25* 1.23* 0.97 1.1 2.23Skeletal Muscle TA 3.30*** 3.45**** 3.95*** 4.23**** 2.80**** 3.34****3.00*** 3.24**** Quad 3.37*** 2.52**** 3.35**** 2.70**** 2.46****2.89*** 2.80**** 2.68*** 2.45*** Gastroc 3.03*** 2.98**** 3.4 3.64****2.49**** 3.22**** 2.92**** 3.25**** 2.82**** Off Target Liver 2.81 2.914.57 3.26 2.73 2.78 3.19 3.78 3.45

1. A peptide comprising at least one directly glycosylated amino acidresidue and one or more arginine-rich arm domains, wherein the totallength of the peptide is 40 amino acid residues or less.
 2. A peptideaccording to claim 1, wherein the at least one directly glycosylatedamino acid residue is O-linked glycosylated, N-linked glycosylated orS-linked glycosylated.
 3. A peptide according to claim 1, wherein atleast one directly glycosylated amino acid residue is glycosylated at afunctional group present in the amino acid side chain selected from anOH, NH₂, NH₃ and SH.
 4. A peptide according to claim 1, wherein the atleast one directly glycosylated amino acid residue is selected from aglycosylated serine, cysteine, threonine, asparagine, glutamine,aminoproline, hydroxyproline, tyrosine, lysine, and amino acid analoguesthereof, preferably the at least one directly glycosylated amino acidresidue is a glycosylated serine. 5-7. (canceled)
 8. A peptide accordingto claim 1, wherein the at least one directly glycosylated amino acidresidue is glycosylated with a sugar selected from: glucose, allose,altrose, idose, gulose, talose, xylose, lactose, mannose, galactose,mannoseamine, glucosamine, galactosamine, N-acetylgalactosamine,D-2-Acetylamino Glucose, N-acetylglucosamine, lactose, maltose,isomaltose, trehalose and sialic acid, preferably the at least one aminoacid residue is directly glucosylated. 9-10. (canceled)
 11. A peptideaccording to claim 8, wherein the at least one amino acid residue isdirectly glucosylated with β-D glucose, preferably the at least oneamino acid residue is a β-D glucosyl serine.
 12. (canceled)
 13. Apeptide according to claim 1, wherein the arginine-rich arm domainscomprise a combined total of between 5-10 Arginine residues.
 14. Apeptide according to claim 1, wherein the arginine-rich arm domainscomprise no more than 3 contiguous Arginine residues.
 15. A peptideaccording to claim 1, wherein the arginine-rich arm domains comprise alength of between 1-12 amino acid residues, preferably wherein the aminoacid residues are selected from the group consisting of: arginine,alanine, beta-alanine, histidine, proline, glycine, cysteine,tryptophan, hydroxyproline, aminohexanoic acid, 3-azetidine-carboxylicacid (Az), 1-(amino)cyclohexanecarboxylic acid (Cy), amino acidanalogues thereof, and any other non-natural amino acid.
 16. (canceled)17. A peptide according to claim 1, wherein each arginine-rich armdomain is selected from the following sequences: RXRRBRRXR (SEQ IDNO.81), RXRBRXR (SEQ ID NO.82), RXRRBRR (SEQ ID NO.83), RBRXR (SEQ IDNO.84), RBRRBRRBR (SEQ ID NO.85), RBRBRBR (SEQ ID NO.86), RGRRGRRGR (SEQID NO.87), RGRGRGR (SEQ ID NO.88), RPRRPRRPR (SEQ ID NO.89), RPRPRPR(SEQ ID NO.90), RHypRRHypRRHypR (SEQ ID NO.91), RHypRHypRHypR (SEQ IDNO.92), RARRARRAR (SEQ ID NO.93), RARARAR (SEQ ID NO.94),RCy*RRCy*RRCy*R (SEQ ID NO.95), RCy*RCy*RCy*R (SEQ ID NO.96), RRBRRBR(SEQ ID NO.97), RBRRBR (SEQ ID NO.98), RRBR (SEQ ID NO.99), RBR, R,RBRBR (SEQ ID NO.100), RBRBRR (SEQ ID NO.101), RBRRR (SEQ ID NO.102),RRRR (SEQ ID NO.103), RBRRBRRR (SEQ ID NO.104, RBRRRRR (SEQ ID NO.105),RRRRRR (SEQ ID NO.106), RRBRR (SEQ ID NO.107), RGRR (SEQ ID NO.108),GRRGR (SEQ ID NO.109), RGGRBRGGR (SEQ ID NO.110), RXRRBRRXRRXRBRXR (SEQID NO.113), RXRR (SEQ ID NO.114, RRXR (SEQ ID NO.115), RXR, RRBRBRXR(SEQ ID NO.117), RRBRRBRBRXR (SEQ ID NO.118), RXRRBRRBR (SEQ ID NO.119),RXRRBRRBRBR (SEQ ID NO.120), RXRRBR (SEQ ID NO.121), RXRBRR (SEQ IDNO.122), HXHRBRRXR (SEQ ID NO.123), RXHBHXR (SEQ ID NO.124), RR, RXRXR(SEQ ID NO.125), BRBRBR (SEQ ID NO.127), BRKBRKRBBR (SEQ ID NO.128),BRKBRKRBBRK (SEQ ID NO.129), RAzRRAzRR (SEQ ID NO.130), RAzRAzR (SEQ IDNO.131), and RXRBR (SEQ ID NO.132).
 18. (canceled)
 19. A peptideaccording to claim 1, wherein each of the arginine-rich arm domainspresent in the peptide is separated from any other arginine rich armdomain present in the peptide by a directly glycosylated amino acidresidue.
 20. A peptide according to claim 1, wherein the peptidecomprises a first arginine-rich arm domain selected from the followingsequences: RXRRBRRXR (SEQ ID NO.81), RXRRBRR (SEQ ID NO.83), RBRBR (SEQID NO.100), and RXRXR (SEQ ID NO.125).
 21. A peptide according to claim20, wherein the peptide comprises a second arginine-rich arm domainselected from the following sequences: RXRBRXR (SEQ ID NO.82), RBRXR(SEQ ID NO.84), RBRRBR (SEQ ID NO.98), RXRBR (SEQ ID NO.132), RBRBR (SEQID NO.100), and RXRXR (SEQ ID NO.125).
 22. A peptide according to claim1, wherein the peptide comprises one or more hydrophobic core domains.23. A peptide according to claim 22, wherein the each hydrophobic coredomain comprises between 1-4 hydrophobic amino acid residues, preferablywherein the amino acid residues are selected from glycine, alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine,tryptophan, and amino acid analogues thereof. 24-25. (canceled)
 26. Apeptide according to claim 22, wherein each hydrophobic core domain iscontiguous with the at least one directly glycosylated amino acidresidue, preferably wherein each hydrophobic core domain contiguous withthe directly glycosylated amino acid residue is positioned between twoflanking arginine-rich arm domains.
 27. (canceled)
 28. A peptideaccording to claim 22, wherein each hydrophobic core domain is selectedfrom the following sequences: GFTGPL (SEQ ID NO.133), QFL, Z, ZL, F, FL,FQILY (SEQ ID NO.134), FQ, WF, QF, FQ, and YQFLI (SEQ ID NO.135). 29-34.(canceled)
 35. A peptide according to claim 1, wherein the peptide isselected from the following sequences: RXRRBRRXRS*FLRXRBRXR (SEQ IDNO.14), RXRRBRRFS*RBRXR (SEQ ID NO.17), RXRRBRRZS*RBRXR (SEQ ID NO.73),RXRRBRRFS1*RBRXR (SEQ ID NO.74), RBRBRS*RBRBR (SEQ ID NO.70) andRXRXRS*RXRXR (SEQ ID NO.71); wherein Z represents1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, and wherein S¹*represents D-serine glycosylated with D-Glucose sugar.
 36. A conjugatecomprising the peptide according to claim 1 covalently linked to atherapeutic molecule.
 37. A conjugate according to claim 36, wherein thetherapeutic molecule is selected from: a nucleic acid, peptide nucleicacid, antisense oligonucleotide (such as PNA, PMO), short interferingRNA, micro RNA, peptide, cyclic peptide, protein, pharmaceutical anddrug, preferably the therapeutic molecule is an antisenseoligonucleotide. 38-39. (canceled)
 40. A method of treating a disease ina subject comprising administering a conjugate according to claim 36 tothe subject in a therapeutically effective amount.
 41. A method oftreating a disease according to claim 40, wherein the disease is of thecentral nervous system.
 42. A method of treating a disease according toclaim 40, wherein the disease is caused by splicing deficiencies.
 43. Amethod of treating a disease according to claim 40, wherein the diseaseis selected from: Duchenne Muscular Dystrophy (DMD), Bucher MuscularDystrophy (BMD), Menkes disease, Beta-thalassemia, dementia, Parkinson'sDisease, Spinal Muscular Atrophy (SMA), myotonic dystrophy (DM),Huntington's Disease, Hutchinson-Gilford Progeria Syndrome,Ataxia-telangiectasia, and cancer. 44-50. (canceled)